CN101441148A - Test method of mizoribine and/or ribavirin - Google Patents
Test method of mizoribine and/or ribavirin Download PDFInfo
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- CN101441148A CN101441148A CN 200810176320 CN200810176320A CN101441148A CN 101441148 A CN101441148 A CN 101441148A CN 200810176320 CN200810176320 CN 200810176320 CN 200810176320 A CN200810176320 A CN 200810176320A CN 101441148 A CN101441148 A CN 101441148A
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Abstract
The subject of the invention is to provide a method for measuring mizoribine and/or ribavirin, in particular provide a simple method for measuring mizoribine and/or ribavirin. wherein a first enzyme capable of catalyzing a first reaction and a second enzyme capable of catalyzing a second reaction are used, the first reaction is a reaction of phosphorylation of mizoribine and/or ribavirin, the second reaction is different from the first reaction, which is not restrained by mizoribine and ribavirin, but restrained by mizoribine phosphate and ribavirin phosphate generated in the first reaction.
Description
Technical field
The present invention relates to the assay method and the phosphorylation method thereof of mizoribine and/or Ribavirin.
Background technology
5-hydroxyl-1-β-D-ribofuranosyl-1H-imidazoles-4-carboxylic acid amides (4-carbamoyl-1-β-D-ribofuranosylimidazolium-5-olate, below note is made mizoribine (Mizoribine) sometimes) get the nod in validity to the aspects such as inhibition of the rejection of kidney transplant, be existing commercially available immunodepressant (patent documentation 1,2).
In addition, mizoribine has been carried out the research (non-patent literature 1) of various mechanism.In addition, as the assay method of existing mizoribine, known have an assay method (non-patent literature 2) that utilizes HPLC, LC/MS/MS.
1-β-D-ribofuranosyl-1H-1,2,4,-triazole-3-carboxylic acid amides (1-β-D-ribofuranosyl-1H-1,2,4-tri-azole-3-carboxamide, below sometimes note make Ribavirin (Ribavirin)) be existing commercially available antiviral agent, when itself and interferon-' alpha ' share, can improve result of treatment to chronic hepatitis C etc.As the assay method of existing Ribavirin, known have an assay method (non-patent literature 3,4) that utilizes HPLC, LC/MS/MS.
In addition, enumerated protein as the preferred embodiment of first enzyme of this part invention hereinafter from Thailand's bulkholderia cepasea (Burkholderia thailandensis), as the information of this protein, disclosing in the non-patent literature 5 may be the base sequence of this protein, the content of ribokinase.
Patent documentation 1:USP3888843
Patent documentation 2:USP5442051
Non-patent literature 1:Pediatr.Int., 44,196-198,2002
Non-patent literature 2:J.Chromato., 222 volumes, 156 pages, 1981
Non-patent literature 3:Yeh L.T. etc., J.Chromatogr.Sci., 41 volumes, 255 pages, 2003
Non-patent literature 4:Yeh L.T. etc., J.Pharm.Biomed.Anal., 43 volumes, 1057 pages, 2007
Non-patent literature 5:BMC Genomics 6,174-187,2005
Summary of the invention
Problem of the present invention provides the straightforward procedure of measuring mizoribine and/or Ribavirin.
In order to solve above-mentioned problem, the inventor furthers investigate repeatedly, new discovery as a result can to mizoribine and/or the acidifying of Ribavirin available phosphorus first the reaction carry out first enzyme of catalysis, thereby finished the phosphorylation method of mizoribine and/or Ribavirin.And then the mizoribine that uses this first enzyme and/or the assay method of Ribavirin have been finished.Promptly, developed utilize this first enzyme and can catalysis the mizoribine of second enzyme of second reaction and/or Ribavirin assay method and measure and use composition, thereby finished the present invention, described second reaction is and the different reaction of described first reaction, it is not subjected to mizoribine and Ribavirin, and the two suppresses, and the two suppresses but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and phosphoric acid Ribavirin.
That is, the present invention relates to following technical proposals.
[0] assay method of mizoribine and/or Ribavirin, it comprises following (1) operation:
(1) comprises the operation of first reaction, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine and/or the Ribavirin that may contain in the sample carried out phosphorylation mizoribine and/or Ribavirin.
[1] as the assay method of [0] described mizoribine and/or Ribavirin, it comprises following (1), (2) and (3) each operation:
(1) comprises the operation of first reaction, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine and/or the Ribavirin that may contain in the sample carried out phosphorylation mizoribine and/or Ribavirin;
(2) suppress second operation of reacting, wherein, can catalysis second reaction second enzyme in the presence of, when carrying out second reaction, upright guest of phosphoimidazole and/or phosphoric acid Ribavirin that above-mentioned first reaction is generated contact with second enzyme, make second reaction be subjected to the inhibition of the degree corresponding with the amount of upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin, and described second reaction is and the different reaction of above-mentioned first reaction, be following<a 〉~<c in any one reaction
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses,
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses,
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin;
(3) detect the operation that second reaction is suppressed degree.
[1-1] as the assay method of [0] or [1] described mizoribine and/or Ribavirin, it comprises each operation in following (1), (2) and (3):
(1) comprises the operation of first reaction, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine and/or the Ribavirin that may contain in the sample carried out phosphorylation mizoribine and/or Ribavirin;
(2) suppress second operation of reacting, wherein, when carrying out second reaction, upright guest of phosphoimidazole and/or phosphoric acid Ribavirin that above-mentioned first reaction is generated contact with second enzyme, make second reaction be subjected to the inhibition of the degree corresponding with the amount of upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin, described second reaction is and the different reaction of first reaction, it is not subjected to mizoribine and Ribavirin, and the two suppresses, and the two suppresses but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and phosphoric acid Ribavirin;
(3) detect the operation that second reaction is suppressed degree.
[1-1-1] as the assay method of [1-1] described mizoribine and/or Ribavirin, wherein, above-mentioned (2) operation is as follows:
(2) suppress second operation of reacting, wherein, can catalysis second reaction second enzyme in the presence of, when carrying out second reaction, upright guest of phosphoimidazole and/or phosphoric acid Ribavirin that above-mentioned first reaction is generated contact with second enzyme, make second reaction be subjected to the inhibition of the degree corresponding with the amount of upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin, and described second reaction is and the different reaction of above-mentioned first reaction, be following<a 〉~<c in any one reaction
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses,
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses,
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin.
[1-2] as each described assay method of [0], [1]~[1-1], wherein, first enzyme is following any one enzyme:
(1) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6; Perhaps
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[1-3] as each described assay method of [0], [1]~[1-2], and wherein, first enzyme is the enzyme that can carry out phosphorylation to mizoribine and/or Ribavirin, and its amino acid sequence by sequence table sequence numbering 1 constitutes.
[1-4] is as each described assay method of [0], [1]~[1-3], wherein, first enzyme is the enzyme that can carry out phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6.
[1-5] as each described assay method of [0], [1]~[1-4], wherein, the base sequence of first enzyme of encoding is the base sequence of sequence table sequence numbering 2 expressions.
[1-6] as each described assay method of [0], [1]~[1-5], wherein, first enzyme has following<1 〉~<4 the enzyme of physicochemical property:
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[1-7] as [1-6] described assay method, wherein, this phosphodonor is ATP.
As each described assay method of [0], [1]~[1-7], wherein, first enzyme belongs to from bulkholderia cepasea (Burkholderia) [1-8].
[1-9] as each described assay method of [0], [1]~[1-8], wherein, first enzyme is from Thailand's bulkholderia cepasea.
[1-10] as each described assay method of [0], [1]~[1-9], wherein, first enzyme is from Thailand bulkholderia cepasea DSM13276 strain.
As each described assay method of [0], [1]~[1-10], wherein, first enzyme is about 34kDa by the molecular weight that the sds polyacrylamide gel electrophoresis method obtains [1-11].
[1-12] as each described assay method of [0], [1]~[1-11], wherein, second enzyme is the enzyme that is subjected to the upright guest of phosphoimidazole to suppress, be subjected to the phosphoric acid Ribavirin to suppress or is subjected to upright guest of phosphoimidazole and the two inhibition of phosphoric acid Ribavirin.
[1-13] as each described assay method of [0], [1]~[1-12], wherein, second enzyme is the enzyme that is subjected to upright guest of phosphoimidazole and the two inhibition of phosphoric acid Ribavirin.
[1-14] as each described assay method of [0], [1]~[1-13], wherein, second enzyme is inosine a 5 ' monophosphate dehydrogenase.
[1-15] as each described assay method of [0], [1]~[1-14], wherein, second enzyme is following any one enzyme:
" 1 " enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, its amino acid sequence by sequence table sequence numbering 7 or sequence table sequence numbering 9 constitutes;
" 2 " enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 11 and the amino acid sequence of sequence table sequence numbering 12; Perhaps
" 3 " have following<i 〉~<v the enzyme of physicochemical property,
<i〉effect
Act on NAD (P) class and inosine 5 ' one phosphoric acid, catalysis generates the reaction of NAD (P) H class and xanthosine 5 ' one phosphoric acid;
<ii〉optimal pH
pH8~9;
<iii〉pH stability
Scope in pH6~11 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<iv〉thermal stability
In the aqueous solution of the 50mM kaliumphosphate buffer pH7 that contains 1mMDTT, when carrying out 30 minutes thermal treatment for 60 ℃, keep the activity more than 85%;
<v〉inhibiting effect
At least upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress its effect.
[1-16] as each described assay method of [0], [1]~[1-15], wherein, second enzyme is also to have following<vi〉the enzyme of physicochemical property:
<vi〉molecular weight
The molecular weight that utilizes the sds polyacrylamide gel electrophoresis method to obtain is 52~54kDa.
[1-17] as each described assay method of [0], [1]~[1-16], and wherein, second enzyme is the enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, and its amino acid sequence by sequence table sequence numbering 7 or sequence table sequence numbering 9 constitutes.
[1-18] is as each described assay method of [0], [1]~[1-17], wherein, second enzyme is the enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 11 and the amino acid sequence of sequence table sequence numbering 12.
[1-19] as each described assay method of [0], [1]~[1-18], wherein, the base sequence of second enzyme of encoding is the base sequence with sequence table sequence numbering 8 or 10 expressions of sequence table sequence numbering.
[1-20] as each described assay method of [0], [1]~[1-19], wherein, second enzyme has following<i 〉~<v the enzyme of physicochemical property:
<i〉effect
Act on NAD (P) class and inosine 5 ' one phosphoric acid, catalysis generates the reaction of NAD (P) H class and xanthosine 5 ' one phosphoric acid;
<ii〉optimal pH
pH8~9;
<iii〉pH stability
Scope in pH6~11 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<iv〉thermal stability
In the aqueous solution of the 50mM kaliumphosphate buffer pH7 that contains 1mM DTT, when carrying out 30 minutes thermal treatment for 60 ℃, keep the activity more than 85%;
<v〉inhibiting effect
At least upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress its effect.
[1-21] as each described assay method of [0], [1]~[1-20], wherein, second enzyme is also to have following<vi〉the enzyme of physicochemical property:
<vi〉molecular weight
The molecular weight that utilizes the sds polyacrylamide gel electrophoresis method to obtain is 52~54kDa.
As each described assay method of [0], [1]~[1-21], wherein, second enzyme belongs to from bacillus (Bacillus) or bacillus marinus (Oceanobacillus) belongs to [1-22].
[1-23] as each described assay method of [0], [1]~[1-22], wherein, second enzyme is from hay bacillus (Bacillus subtilis) or Oceanobacillus iheyensis.
[1-24] as each described assay method of [0], [1]~[1-23], wherein, second enzyme is from hay bacillus ATCC23857 strain or Oceanobacillus iheyensis DSM14731 strain.
[1-25] as each described assay method of [0], [1]~[1-24], wherein, (1) operation is the operation that also contains phosphodonor.
[1-26] as each described assay method of [0], [1]~[1-25], wherein, the operation of (1) is the operation that also contains metallic ion.
[1-27] as [1-26] described assay method, wherein, this phosphodonor is ATP.
[1-28] as [1-27] described assay method, and wherein, metallic ion is more than in magnesium ion, cobalt ions, nickel ion and the manganese ion any one.
[1-29] as each described assay method of [0], [1]~[1-28], wherein, (2) operation is the operation that also contains inosine 5 ' one phosphoric acid.
[1-30] as each described assay method of [0], [1]~[1-29], wherein, (2) operation is the operation that also contains NAD (P) class.
[1-31] as each described assay method of [0], [1]~[1-30], wherein, upright guest of this phosphoimidazole and phosphoric acid Ribavirin are the upright guest of single phosphoimidazole and single phosphoric acid Ribavirin.
[1-32] as each described assay method of [0], [1]~[1-31], wherein, the detection that suppresses degree in (3) operation is an operation of measuring second enzyme reaction rate.
[1-33] as each described assay method of [0], [1]~[1-32], wherein, the detection that suppresses degree in (3) operation is an operation of measuring the variable quantity of NAD (P) class.
[1-34] is as each described assay method of [0], [1]~[1-33], wherein, (3) detection that suppresses degree in the operation is following operation: the mizoribine and/or the caused variable quantity of Ribavirin of the known quantity after operation (1) and (2) are handled and the caused variable quantity of determination object detects and comparison, thus the mizoribine and/or the Ribavirin that may contain in the sample carried out quantitatively.
[2] assay method of mizoribine, wherein, described method comprises following (1), (2) and (3) each operation:
(1) comprises first operation of reacting, in described first reaction, in the presence of the enzyme that can carry out phosphorylation, the mizoribine that may contain in the sample is carried out phosphorylation mizoribine;
(2) suppress second operation of reacting, wherein, can catalysis this second reaction second enzyme in the presence of, when carrying out second reaction, the upright guest of this phosphoimidazole that above-mentioned first reaction is generated contacts with second enzyme, makes second reaction be subjected to the inhibition of the degree corresponding with the upright guest's of this phosphoimidazole amount, and described second reaction is and the different reaction of above-mentioned first reaction, it is not suppressed by mizoribine, but the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses; With
(3) detect the operation that second reaction is suppressed degree.
[2-1] as the assay method of above-mentioned [2] described mizoribine, wherein, described method has any one feature at least of record among above-mentioned [0], [1]~[1-34].
[3] assay method of Ribavirin, wherein, described method comprises following (1), (2) and (3) each operation:
(1) comprises first operation of reacting, in described first reaction, in the presence of the enzyme that can carry out phosphorylation, the Ribavirin that may contain in the sample is carried out phosphorylation Ribavirin;
(2) suppress second operation of reacting, wherein, can catalysis this second reaction second enzyme in the presence of, when carrying out second reaction, this phosphoric acid Ribavirin that above-mentioned first reaction is generated contacts with second enzyme, makes second reaction be subjected to the inhibition of the degree corresponding with the amount of this phosphoric acid Ribavirin, and described second reaction is and the different reaction of above-mentioned first reaction, it is not suppressed by Ribavirin, but the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses; With
(3) detect the operation that second reaction is suppressed degree.
[3-1] as the assay method of above-mentioned [3] described Ribavirin, wherein, described method has any one feature at least of record among above-mentioned [0], [1]~[1-34].
[4] assay method of mizoribine and/or Ribavirin, it comprises each operation in following (a)~(c):
(a) comprise the operation of reacting I, in described reaction I, in following (1)~(3) arbitrarily enzyme and ATP in the presence of, make the mizoribine and/or the Ribavirin that may contain in the sample become the upright guest of phosphoimidazole and/or phosphoric acid Ribavirin and ADP;
(1) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes,
(2) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its by disappearance in the amino acid sequence of sequence table sequence numbering 1, replace or the amino acid whose amino acid sequence that added one or more constitutes, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of ATP, catalysis makes mizoribine and/or Ribavirin become the reaction of the upright guest of phosphoimidazole and/or phosphoric acid Ribavirin and ADP; Perhaps,
At least in the presence of ATP, catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%;
(b) in the presence of the enzyme B of catalysis reaction II different or plural reaction II or plural enzyme B with above-mentioned reaction I, the operation that the amount of the ADP that the corresponding above-mentioned reaction I of the determination object that reaction II is produced produces changes;
(c) detect the variable quantity of this determination object, measure the operation of the amount of the mizoribine that may contain in the sample and/or Ribavirin.
[4-1] as the assay method of [4] described mizoribine and/or Ribavirin, wherein, upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin are the upright guest of single phosphoimidazole and/or single phosphoric acid Ribavirin.
As the assay method of each described mizoribine of [4]~[4-1] and/or Ribavirin, wherein, (a) the described enzyme in the operation is the enzyme that is made of sequence table sequence numbering 1 described amino acid sequence [4-2].
[4-3] is as the assay method of each described mizoribine of [4]~[4-2] and/or Ribavirin, wherein, (a) the described enzyme in the operation is by disappearance in the amino acid sequence of sequence table sequence numbering 1, replaces or added the enzyme that one or more amino acid whose amino acid sequence constitutes.
[4-4] is as the assay method of each described mizoribine of [4]~[4-3] and/or Ribavirin, wherein, (a) the described enzyme in the operation is the enzyme that can carry out phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or more amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6.
[4-5] as the assay method of each described mizoribine of [4]~[4-4] and/or Ribavirin, wherein, (a) the described enzyme in the operation has following<1 〉~<4 the enzyme of physicochemical property:
<1〉effect
At least in the presence of ATP, catalysis makes mizoribine and/or Ribavirin become the reaction of the upright guest of phosphoimidazole and/or phosphoric acid Ribavirin and ADP; Perhaps,
At least in the presence of ATP, catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[4-6] as the assay method of each described mizoribine of [4]~[4-5] and/or Ribavirin, wherein, (a) the described enzyme in the operation is from Thailand's bulkholderia cepasea.
[4-7] as the assay method of each described mizoribine of [4]~[4-6] and/or Ribavirin, wherein, (a) the described enzyme in the operation is from Thailand bulkholderia cepasea DSM13276 strain.
[4-8] as the assay method of each described mizoribine of [4]~[4-7] and/or Ribavirin, wherein, (a) the described enzyme in the operation utilizes magnesium ion, cobalt ions, nickel ion or manganese ion at least.
[4-9] as the assay method of each described mizoribine of [4]~[4-8] and/or Ribavirin, wherein,
(b) comprise (b-1) reaction and (b-2) reaction in the operation, (b-1) in the reaction, in the presence of ADP dependence hexokinase, make glucose and ADP become G-6-P and AMP; (b-2) in the reaction, in the presence of glucose-6-phosphate dehydrogenase (G6PD), the G-6-P that NAD (P) class and above-mentioned reaction are generated becomes NAD (P) H class and gluconolactone-6-phosphoric acid; The amount of the ADP that the corresponding above-mentioned reaction I of NAD (P) H class generates increases,
(c) operation is to detect the operation of the recruitment of NAD (P) H class.
[4-10] as the assay method of each described mizoribine of [4]~[4-9] and/or Ribavirin, wherein,
(b) comprise (b-1) reaction and (b-2) reaction in the operation, (b-1) in the reaction, in the presence of pyruvate kinase, make ADP and phosphoenolpyruvate (this instructions remember sometimes make PEP) become ATP and pyruvic acid; (b-2) in the reaction, in the presence of lactic dehydrogenase, the pyruvic acid that NAD (P) H class and above-mentioned reaction are generated becomes NAD (P) class and lactic acid; The amount of the ADP that the corresponding above-mentioned reaction I of NAD (P) H class generates reduces,
(c) operation is to detect the operation of the reduction of NAD (P) H class.
[4-11] as the assay method of each described mizoribine of [4]~[4-10] and/or Ribavirin, wherein,
(b) comprise (b-1) reaction and (b-2) reaction in the operation, (b-1) in the reaction, in the presence of pyruvate kinase, make ADP and PEP become pyruvic acid and ATP; (b-2) in the reaction, in the presence of pyruvate oxidase, the pyruvic acid that oxygen, phosphoric acid and above-mentioned reaction are generated becomes hydrogen peroxide and acetyl phosphate; The amount of the ADP that the corresponding above-mentioned reaction I of hydrogen peroxide generates increases,
(c) operation is the operation with the amount of detection hydrogen peroxide such as hydrogen peroxide indicator.
[4-12] is as the assay method of each described mizoribine and/or Ribavirin among [4]~[4-11], wherein, (c) detection of variable quantity is following operation in the operation: the mizoribine and/or the caused variable quantity of Ribavirin of the known quantity after to operation (a) and (b) handling and the caused variable quantity of determination object detects and relatively, thus the mizoribine and/or the Ribavirin that may contain in the sample carried out quantitatively.
[4-13] as the assay method of any described mizoribine and/or Ribavirin among [4]~[4-12], wherein, the molecular weight that this enzyme utilizes the sds polyacrylamide gel electrophoresis method to obtain is about 34kDa.
[5] assay method of mizoribine, this method comprise following (a)~(c) each operation.
(a) comprise the operation of reacting I, in described reaction I, in following (1)~(3) arbitrarily enzyme and ATP in the presence of, make the mizoribine that may contain in the sample become upright guest of phosphoimidazole and ADP;
(1) can carry out the enzyme of phosphorylation to mizoribine, its amino acid sequence by sequence table sequence numbering 1 constitutes,
(2) can carry out the enzyme of phosphorylation to mizoribine, it is to constitute by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or more amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of ATP, catalysis makes mizoribine become the reaction of upright guest of phosphoimidazole and ADP;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%;
(b) in the presence of the enzyme B of catalysis reaction II different or plural reaction II or plural enzyme B with above-mentioned reaction I, the operation that the amount of the ADP that the corresponding above-mentioned reaction I of the determination object that reaction II is produced produces changes;
(c) detect the variable quantity of this determination object, measure the operation of the amount of the mizoribine that may contain in the sample.
[5-1] as above-mentioned [5] described assay method, wherein, described method has any at least feature of record among above-mentioned [4]~[4-13].
[6] assay method of Ribavirin, this method comprise each operation in following (a)~(c).
(a) comprise the operation of reacting I, in described reaction I, in following (1)~(3) arbitrarily enzyme and ATP in the presence of, make the Ribavirin that may contain in the sample become phosphoric acid Ribavirin and ADP;
(1) can carry out the enzyme of phosphorylation to Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes,
(2) can carry out the enzyme of phosphorylation to Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or add one or more amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of ATP, catalysis makes Ribavirin become the reaction of phosphoric acid Ribavirin and ADP;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%;
(b) in the presence of the enzyme B of catalysis reaction II different or plural reaction II or plural enzyme B with above-mentioned reaction I, the operation that the amount of the ADP that the corresponding above-mentioned reaction I of the determination object that reaction II is produced produces changes;
(c) detect the variable quantity of this determination object, measure the operation of the amount of the Ribavirin that may contain in the sample.
[6-1] as above-mentioned [6] described assay method, wherein, described method has any at least feature of record among above-mentioned [4]~[4-13].
[7] the phosphorylation method of mizoribine and/or Ribavirin, this method is used any one enzyme in following (1)~(3):
(1) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[7-1] as [7] described phosphorylation method, wherein, this phosphodonor is ATP.
[7-2] as each described phosphorylation method of [7]~[7-1], wherein, upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin are the upright guest of single phosphoimidazole and/or single phosphoric acid Ribavirin.
[7-3] as each described phosphorylation method of [7]~[7-2], wherein, can carry out the molecular weight that the enzyme of phosphorylation utilizes the sds polyacrylamide gel electrophoresis method to obtain to mizoribine and/or Ribavirin and be about 34kDa.
[7-4] wherein, uses the enzyme that is made of the amino acid sequence of putting down in writing in the sequence table sequence numbering 1 as each described phosphorylation method of [7]~[7-3].
[7-5] is as each described phosphorylation method of [7]~[7-4], wherein, use can be carried out the enzyme of phosphorylation to mizoribine and/or Ribavirin, this enzyme constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6.
[7-6] as each described phosphorylation method of [7]~[7-5], wherein, the base sequence of this enzyme of encoding is the base sequence with 2 expressions of sequence table sequence numbering.
[7-7] as each described phosphorylation method of [7]~[7-6], wherein, uses to have following<1 〉~<4 the enzyme of physicochemical property:
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[7-8] as [7-7] described phosphorylation method, wherein, this phosphodonor is ATP.
[7-9] as each described phosphorylation method of [7]~[7-8], wherein, the molecular weight that this enzyme utilizes the sds polyacrylamide gel electrophoresis method to obtain is about 34kDa.
As each described phosphorylation method of [7]~[7-9], wherein, this enzyme belongs to from bulkholderia cepasea [7-10].
[7-11] as each described phosphorylation method of [7]~[7-10], wherein, this enzyme is from Thailand's bulkholderia cepasea.
[7-12] as each described phosphorylation method of [7]~[7-11], wherein, this enzyme is from Thailand bulkholderia cepasea DSM13276 strain.
[8] the phosphorylation method of mizoribine, this method is used any one enzyme in following (1)~(3):
(1) can carry out the enzyme of phosphorylation to mizoribine, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of phosphodonor, catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[8-1] as the phosphorylation method of above-mentioned [8] described mizoribine, wherein, described method has any at least feature of above-mentioned [7]~[7-12] record.
[9] the phosphorylation method of Ribavirin, this method is used any one enzyme in following (1)~(3):
(1) can carry out the enzyme of phosphorylation to Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of phosphodonor, catalysis makes Ribavirin become the reaction of phosphoric acid Ribavirin;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[9-1] as the phosphorylation method of above-mentioned [9] described Ribavirin, wherein, described method has any at least feature of record among above-mentioned [7]~[7-12].
[10] manufacture method of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin wherein, is used any one enzyme in following (1)~(3), makes mizoribine and/or Ribavirin become upright guest of phosphoimidazole and/or phosphoric acid Ribavirin;
(1) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[10-1] as above-mentioned [10] described manufacture method, wherein, described method has any at least feature of above-mentioned [7]~[7-12] record.
[11] the upright guest's of phosphoimidazole manufacture method wherein, is used any one enzyme in following (1)~(3), makes mizoribine become the upright guest of phosphoimidazole;
(1) can carry out the enzyme of phosphorylation to mizoribine, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of phosphodonor, catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[11-1] as above-mentioned [11] described manufacture method, wherein, described method has any at least feature of above-mentioned [7]~[7-12] record.
[12] manufacture method of phosphoric acid Ribavirin wherein, is used any one enzyme in following (1)~(3), makes Ribavirin become the phosphoric acid Ribavirin;
(1) can carry out the enzyme of phosphorylation to Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of phosphodonor, catalysis makes Ribavirin become the reaction of phosphoric acid Ribavirin;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[12-1] as above-mentioned [12] described manufacture method, wherein, described method has any at least feature of above-mentioned [7]~[7-12] record.
[13] a kind of composition, it contains following compositions:
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to mizoribine and/or Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, its can catalysis second reaction, described second reaction is and the different reaction of above-mentioned first reaction, is following<a 〉~<c in any one reaction,
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses,
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses,
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
[13-0-1] as [13] described composition, wherein, it is used for the mensuration of mizoribine and/or Ribavirin.
[13-0-2] mizoribine and/or Ribavirin are measured and are used composition, and it contains following (A) and (B):
(A) the 1st reagent, it contains following (A1)~(A4) at least:
(A1) first enzyme of effective dose, it can catalysis carry out first reaction of phosphorylation to mizoribine and/or Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4) any one or two compositions in second enzyme of effective dose, inosine 5 ' one phosphoric acid and NAD (P) class, described second enzymatic and the different reaction of first reaction, it is suppressed by upright guest of phosphoimidazole and/or phosphoric acid Ribavirin;
(B) the 2nd reagent, it contains the composition that do not contain in the 1st reagent among second enzyme, inosine 5 ' one phosphoric acid and NAD (P) class that upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress of being subjected to of effective dose at least.
[13-0-3] as [13] described composition, it is the mizoribine mensuration composition that contains following compositions:
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to mizoribine;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, second reaction that its catalysis is different with above-mentioned first reaction, it is not suppressed by mizoribine, but suppressed by the upright guest of phosphoimidazole;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
[13-0-4] as [13] described composition, it is the Ribavirin mensuration composition that contains following compositions:
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, second reaction that its catalysis is different with above-mentioned first reaction, it is not suppressed by Ribavirin, but suppressed by the phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
[13-0-5] as [13] described composition, and it is that the upright guest of phosphoimidazole of containing following compositions measures and uses composition:
(A4-1) second enzyme, second reaction that its catalysis is different with above-mentioned first reaction is suppressed by the upright guest of phosphoimidazole;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
[13-0-6] as [13] described composition, it is the phosphoric acid Ribavirin mensuration composition that contains following compositions:
(A4-1) second enzyme, second reaction that its catalysis is different with above-mentioned first reaction is suppressed by the phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
[13-0-7] wherein, measures with in the composition at this as each described composition of [13]~[13-0-6], adds before being about to mensuration more than any one among following 3 kinds of compositions:
(A4-1) second enzyme;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
[13-1] as [13] described composition, wherein, this phosphodonor is ATP.
[13-2] as the described composition of [13]~[13-1], and wherein, metallic ion is more than among magnesium ion, cobalt ions, nickel ion and the manganese ion any one.
[13-3] mizoribine and/or Ribavirin are measured and are used composition, and it contains following (A) and (B):
(A) the 1st reagent, it contains following (A1)~(A4) at least:
(A1) first enzyme of effective dose, it can carry out phosphorylation to mizoribine and/or Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4) second enzyme of effective dose and NAD (P) class, described second enzyme are suppressed by upright guest of phosphoimidazole and/or phosphoric acid Ribavirin;
(B) the 2nd reagent, it contains inosine 5 ' one phosphoric acid of effective dose at least.
[13-4] as [13-3] described composition, wherein, this phosphodonor is ATP.
[13-5] as each described composition among [13-3]~[13-4], and wherein, metallic ion is more than among magnesium ion, cobalt ions, nickel ion and the manganese ion any one.
[13-6] as each described composition of [13]~[13-5], it further contains following (C):
(C) standard reagent, it contains the mizoribine and/or the Ribavirin of known quantity at least.
[13-7] as each described composition of [13]~[13-6], wherein, first enzyme is any one enzyme in the following enzyme:
(1) enzyme that constitutes by the amino acid sequence of sequence table sequence numbering 1;
(2) by lacking in the amino acid sequence of sequence table sequence numbering 1, replace or having added the enzyme that one or several amino acid whose amino acid sequence constitutes, this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6; Perhaps
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[13-8] as each described composition of [13]~[13-7], and wherein, first enzyme is the enzyme that the amino acid sequence by sequence table sequence numbering 1 constitutes.
[13-9] is as each described composition of [13]~[13-8], wherein, first enzyme is by disappearance in the amino acid sequence of sequence table sequence numbering 1, replaces or added the enzyme that one or several amino acid whose amino acid sequence constitutes, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6.
[13-10] as each described composition of [13]~[13-9], wherein, the base sequence of first enzyme of encoding is the base sequence of sequence table sequence numbering 2 expressions.
[13-11] as each described composition of [13]~[13-10], wherein, first enzyme has following<1 〉~<4 the enzyme of physicochemical property:
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[13-12] as [13-11] described composition, wherein, this phosphodonor is ATP.
[13-13] as each described composition of [13-11]~[13-12], wherein, upright guest of this phosphoimidazole and phosphoric acid Ribavirin are the upright guest of single phosphoimidazole and single phosphoric acid Ribavirin.
As each described composition of [13]~[13-13], wherein, first enzyme belongs to from bulkholderia cepasea [13-14].
[13-15] as each described composition of [13]~[13-14], wherein, first enzyme is from Thailand's bulkholderia cepasea.
[13-16] as each described composition of [13]~[13-15], wherein, first enzyme is from Thailand bulkholderia cepasea DSM13276 strain.
[13-17] as each described composition of [13]~[13-16], wherein, second enzyme is the enzyme that is subjected to the upright guest of phosphoimidazole to suppress, be subjected to the phosphoric acid Ribavirin to suppress or is subjected to upright guest of phosphoimidazole and the two inhibition of phosphoric acid Ribavirin.
[13-18] as each described composition of [13]~[13-17], wherein, second enzyme is the enzyme that is subjected to upright guest of phosphoimidazole and the two inhibition of phosphoric acid Ribavirin.
[13-19] as each described composition of [13]~[13-18], wherein, second enzyme is inosine a 5 ' monophosphate dehydrogenase.
[13-20] as each described composition of [13]~[13-19], wherein, second enzyme is following any one enzyme:
" 1 " enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, its amino acid sequence by sequence table sequence numbering 7 or sequence table sequence numbering 9 constitutes;
" 2 " enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 11 and the amino acid sequence of sequence table sequence numbering 12: perhaps
" 3 " have following<i 〉~<v the enzyme of physicochemical property,
<i〉effect
Act on NAD (P) class and inosine 5 ' one phosphoric acid, catalysis generates the reaction of NAD (P) H class and xanthosine 5 ' one phosphoric acid;
<ii〉optimal pH
pH8~9;
<iii〉pH stability
Scope in pH6~11 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<iv〉thermal stability
In the aqueous solution of the 50mM kaliumphosphate buffer pH7 that contains 1mMDTT, when carrying out 30 minutes thermal treatment for 60 ℃, keep the activity more than 85%;
<v〉inhibiting effect
At least upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress its effect.
[13-21] as each described composition of [13]~[13-20], wherein, second enzyme is also to have following<vi〉the enzyme of physicochemical property:
<vi〉molecular weight
The molecular weight that utilizes the sds polyacrylamide gel electrophoresis method to obtain is 52~54kDa.
[13-22] as each described composition of [13]~[13-21], and wherein, second enzyme is the enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, and its amino acid sequence by sequence table sequence numbering 7 or sequence table sequence numbering 9 constitutes.
[13-23] is as each described composition of [13]~[13-22], wherein, second enzyme is the enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 11 and the amino acid sequence of sequence table sequence numbering 12.
[13-24] as each described composition of [13]~[13-23], wherein, the base sequence of second enzyme of encoding is the base sequence with sequence table sequence numbering 8 or 10 expressions of sequence table sequence numbering.
[13-25] as each described composition of [13]~[13-24], wherein, second enzyme has following<i 〉~<v the enzyme of physicochemical property:
<i〉effect
Act on NAD (P) class and inosine 5 ' one phosphoric acid, catalysis generates the reaction of NAD (P) H class and xanthosine 5 ' one phosphoric acid;
<ii〉optimal pH
pH8~9;
<iii〉pH stability
Scope in pH6~11 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<iv〉thermal stability
In the aqueous solution of the 50mM kaliumphosphate buffer pH7 that contains 1mMDTT, when carrying out 30 minutes thermal treatment for 60 ℃, keep the activity more than 85%;
<v〉inhibiting effect
At least upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress its effect.
[13-26] as each described composition of [13]~[13-25], wherein, second enzyme is also to have following<vi〉the enzyme of physicochemical property:
<vi〉molecular weight
The molecular weight that utilizes the sds polyacrylamide gel electrophoresis method to obtain is 52~54kDa.
As each described composition of [13]~[13-26], wherein, second enzyme belongs to from bacillus or bacillus marinus [13-27].
[13-28] as each described composition of [13]~[13-27], wherein, second enzyme is from hay bacillus or Oceanobacillus iheyensis.
[13-29] as each described composition of [13]~[13-28], wherein, second enzyme is from hay bacillus ATCC23857 strain or Oceanobacillus iheyensis DSM14731 strain.
[14] manufacture method of enzyme, the manufacture method of any one enzyme in its (1)~(3) that are following, described method comprise that the base sequence according to this enzyme of coding forms the operation of this enzyme and obtains the operation of this enzyme;
(1) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes:
(2) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or more amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
As [14] described manufacture method, wherein, the operation that forms this enzyme comprises the step of the cell that uses the base sequence that contains this enzyme of encoding [14-1].
[14-2] as each described manufacture method of [14]~[14-1], wherein, the operation that forms this enzyme comprises the step of using the transformant that imports the base sequence that this enzyme of coding is arranged.
[14-3] as each described manufacture method of [14]~[14-2], wherein, the operation that forms this enzyme comprises the step of the microorganism of using the base sequence with this enzyme of coding.
[15] as each described manufacture method of [14]~[14-3], wherein, the operation that forms this enzyme comprises the step of using the microorganism that belongs to the bulkholderia cepasea genus, and described microorganisms can catalysis make mizoribine and/or Ribavirin become the enzyme of the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin.
[15-1] as each described manufacture method of [14]~[15], wherein, the operation that forms this enzyme comprises the step of using Thailand's bulkholderia cepasea.
[15-2] as each described manufacture method of [14]~[15-1], wherein, the operation that forms this enzyme comprises the step of using Thailand bulkholderia cepasea DSM13276 strain.
[16] as each described manufacture method of [14]~[15-2], wherein, this enzyme is the enzyme that can carry out phosphorylation to mizoribine and/or Ribavirin, and its amino acid sequence by sequence table sequence numbering 1 constitutes.
[16-1] is as each described manufacture method of [14]~[16], wherein, this enzyme is the enzyme that can carry out phosphorylation to mizoribine and/or Ribavirin, its by disappearance in the amino acid sequence of sequence table sequence numbering 1, replace or added one or more amino acid whose amino acid sequence and constitute.
[16-2] is as each described manufacture method of [14]~[16-1], wherein, this enzyme is the enzyme that can carry out phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or more amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6.
[17], wherein, obtain when the base sequence according to this enzyme of coding forms in the operation of this enzyme formed this enzyme, to comprise that further refining back obtains the operation of enzyme as each described manufacture method of [14]~[16-2].
As each described manufacture method of [14]~[17], wherein, the operation that forms this enzyme comprises the step of the cell that uses the base sequence that contains this enzyme of encoding [17-1].
[17-2] as each described manufacture method of [14]~[17-1], wherein, the operation that forms this enzyme comprises the step of using the transformant that imports the base sequence that this enzyme of coding is arranged.
[18] as each described manufacture method of [14]~[17-2], wherein, the base sequence of this enzyme of encoding is the base sequence of sequence table sequence numbering 2 expressions.
[21] as each described manufacture method of [14]~[18], wherein, this enzyme has following<1 〉~<4 the enzyme of physicochemical property:
<1〉effect
At least catalysis makes mizoribine and/or Ribavirin become the reaction of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the presence of phosphodonor; Perhaps,
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
[19-1] as [19] described manufacture method, wherein, upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin are the upright guest of single phosphoimidazole and/or single phosphoric acid Ribavirin.
[19-2] as each described manufacture method of [19]~[19-1], wherein, this phosphodonor is ATP.
[19-3] as each described manufacture method of [14]~[19-2], wherein, the molecular weight that this enzyme utilizes the sds polyacrylamide gel electrophoresis method to obtain is about 34kDa.
[20] a kind of enzyme manufacture method, the manufacture method of any one enzyme in its " 1 "~" 3 " that are following, described method comprise that the base sequence according to this enzyme of coding forms the operation of this enzyme and obtains the operation of this enzyme;
" 1 " enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, its amino acid sequence by sequence table sequence numbering 9 constitutes;
" 2 " enzyme that can carry out oxidation to inosine 5 ' one phosphoric acid, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 9, replacing or added one or more amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 11 and the amino acid sequence of sequence table sequence numbering 12;
" 3 " have following<i 〉~<v the enzyme of physicochemical property,
<i〉effect
Act on NAD (P) class and inosine 5 ' one phosphoric acid, catalysis generates the reaction of NAD (P) H class and xanthosine 5 ' one phosphoric acid;
<ii〉optimal pH
pH8~9;
<iii〉pH stability
Scope in pH6~11 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<iv〉thermal stability
In the aqueous solution of the 50mM kaliumphosphate buffer pH7 that contains 1mM DTT, when carrying out 30 minutes thermal treatment for 60 ℃, keep the activity more than 85%;
<v〉inhibiting effect
At least upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress its effect.
[20-1] as [20] described manufacture method, and wherein, the enzyme that limits in " 3 " is also to have following<vi〉the enzyme of physicochemical property:
<vi〉molecular weight
The molecular weight that utilizes the sds polyacrylamide gel electrophoresis method to obtain is 52~54kDa.
As each described manufacture method of [20]~[20-1], wherein, the operation that forms this enzyme comprises the step of the cell that uses the base sequence that contains this enzyme of encoding [20-2].
[20-3] as each described manufacture method of [20]~[20-2], wherein, the operation that forms this enzyme comprises the step of using the transformant that imports the base sequence that this enzyme of coding is arranged.
[20-4] as each described manufacture method of [20]~[20-3], wherein, the operation that forms this enzyme comprises the step of the microorganism of using the base sequence with this enzyme of coding.
[20-5] as each described manufacture method of [20]~[20-4], wherein, the operation that forms this enzyme comprises the step of using the microorganism that belongs to the bacillus marinus genus.
[20-6] as each described manufacture method of [20]~[20-5], wherein, the operation that forms this enzyme comprises the step of using Oceanobacillus iheyensis strain.
[20-7] as each described manufacture method of [20]~[20-6], wherein, the operation that forms this enzyme comprises the step of using Oceanobacillus iheyensis DSM14731 strain.
[20-8] as each described manufacture method of [20]~[20-7], and wherein, when obtaining in the operation that base sequence according to this enzyme of coding forms this enzyme formed this enzyme, refining back obtains this enzyme.
As each described manufacture method of [20]~[20-8], wherein, the operation that forms this enzyme comprises the step of the cell that uses the base sequence that contains this enzyme of encoding [20-9].
[20-10] as each described manufacture method of [20]~[20-9], wherein, the operation that forms this enzyme comprises the step of using the transformant that imports the base sequence that this enzyme of coding is arranged.
[21] as each described manufacture method of [20]~[20-10], wherein, the base sequence of this enzyme of encoding is the base sequence of sequence table sequence numbering 10 expressions.
[22] assay method of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin, this method comprises following " 1 " and " 2 " each operation:
" 1 " suppresses the operation of second reaction, wherein, at can catalysis following<A 〉~<C in any second reaction second enzyme in the presence of, upright guest of the phosphoimidazole that may contain in the sample and/or phosphoric acid Ribavirin are contacted with second enzyme, make second reaction be subjected to the inhibition of the degree corresponding with the amount of upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin
<A〉be subjected to reaction that the upright guest of the phosphoimidazole that may contain in the sample suppresses,
<B〉be subjected to reaction that the phosphoric acid Ribavirin that may contain in the sample suppresses,
<C〉be subjected to the reaction of upright guest of the phosphoimidazole that may contain in the sample and the two inhibition of phosphoric acid Ribavirin;
" 2 " detect the operation that second reaction is suppressed degree.
[22-1] as above-mentioned [22] described assay method, wherein, second enzyme has any at least feature of record among above-mentioned [1-12]~[1-22].
[22-2] as the described assay method of above-mentioned [22]~[22-1], and wherein, " 1 " operation has any one feature at least that above-mentioned [1-29]~[1-30] puts down in writing in each.
[22-3] as the described assay method of above-mentioned [22]~[22-2], wherein, upright guest of this phosphoimidazole and phosphoric acid Ribavirin are the upright guest of single phosphoimidazole and single phosphoric acid Ribavirin.
[22-4] as the described assay method of above-mentioned [22]~[22-3], and wherein, the detection that suppresses degree in " 2 " operation has any at least feature that above-mentioned [1-32]~[1-33] puts down in writing in each.
[22-5] is as each described assay method of [22]~[22-4], wherein, the detection of the inhibition degree in " 2 " operation is following operation: to the phosphoimidazole of known quantity upright guest and/or the caused variable quantity of phosphoric acid Ribavirin with the caused variable quantity of determination object detects and relatively, thereby upright guest of the phosphoimidazole that may contain in the sample and/or phosphoric acid Ribavirin are carried out quantitatively.
According to the present invention, can provide the straightforward procedure of measuring mizoribine and/or Ribavirin at least.
Description of drawings
Fig. 1 (A) shows the figure that cultivates the SDS-PAGE that transforms the resulting crude enzyme liquid of Rhodococcus erythropolis (Rhodococcus erythropolis) that pTip QC1/BthNK is arranged.Fig. 1 (B) shows the figure that cultivates the SDS-PAGE that transforms the resulting crude enzyme liquid of Rhodococcus erythropolis that pTip QC2/BthNK is arranged.
Fig. 2 is the figure that show to cultivate transforms the SDS-PAGE of the crude enzyme liquid that e. coli bl21 (DE3) that pET21a (+)/BthNK is arranged obtains.
Fig. 3 (A) is the chromatogram that obtains with the upright guest of HPLC separation criterion product phosphoimidazole.Fig. 3 (B) is the chromatogram that obtains with the reactant liquor before the reaction of HPLC separation embodiment 14.Fig. 3 (C) separates the chromatogram that the reacted reactant liquor of embodiment 14 obtains with HPLC.
Fig. 4 is the figure of the optimal pH of explanation BthNK.
Fig. 5 is the figure of the pH stability of explanation BthNK.
Fig. 6 is the figure of the thermal stability of explanation BthNK.
Fig. 7 is the figure that shows the SDS-PAGE of BthNK.
Fig. 8 is the figure that shows the operative temperature scope of BthNK.
Fig. 9 is the Arrhenius figure that shows the operative temperature scope of BthNK.
Figure 10 (A) is the chromatogram that obtains with HPLC separation criterion product ATP, ADP, AMP and adenosine potpourri.Figure 10 (B) is the preceding chromatogram of phosphorylation method of implementing embodiment 23 described adenosines.Figure 10 (C) is the chromatogram of implementing after the phosphorylation method of embodiment 23 described adenosines.
Figure 11 is the figure that shows the calibration curve that utilizes the composition measuring adenosine, inosine and the guanosine potpourri that have used BthNK.
Figure 12 is the figure that shows the calibration curve that utilizes the composition measuring adenosine that has used BthNK.
Figure 13 is the figure that shows the calibration curve that utilizes the composition measuring inosine that has used BthNK.
Figure 14 is the figure that shows the calibration curve that utilizes the composition measuring guanosine that has used BthNK.
Figure 15 is the figure that shows the calibration curve that utilizes the composition measuring adenosine that has used BthNK.
Figure 16 is the figure that shows the calibration curve that utilizes the composition measuring inosine that has used BthNK.
Figure 17 is the figure that shows the calibration curve that utilizes the composition measuring guanosine that has used BthNK.
Figure 18 is the figure of the optimal pH of explanation BsuIMPDH (A) and ObIMPDH (B).
Figure 19 is the figure of the pH stability of explanation BsuIMPDH (A) and ObIMPDH (B).
Figure 20 is the figure of the thermal stability of explanation BsuIMPDH (A) and ObIMPDH (B).
Figure 21 is the figure that shows the SDS-PAGE of BsuIMPDH (B) and ObIMPDH (A).
Figure 22 is explanation KCl (zero) and ammonium chloride (●) figure to the influence of the activity of BsuIMPDH (B) and ObIMPDH (A).
Figure 23 is the figure of calibration curve of composition measuring mizoribine that show to utilize the BsuIMPDH of first enzyme that used embodiment 2 preparations and embodiment 33 preparations.
Figure 24 is the figure of calibration curve of composition measuring Ribavirin that show to utilize the BsuIMPDH of the BthNK that used embodiment 12 preparations and embodiment 33 preparations.
Figure 25 is the figure of calibration curve of composition measuring mizoribine that show to utilize the BsuIMPDH of the BthNK that used embodiment 11 preparations and embodiment 33 preparations.
Figure 26 be show to utilize the BthNK that used embodiment 11 preparations and embodiment 33 preparations the composition measuring of BsuIMPDH with the calibration curve (zero) of the mizoribine of distilled water diluting with measure the figure of the calibration curve (●) of the mizoribine that dilutes with serum.
Figure 27 is the figure of calibration curve of composition measuring mizoribine that show to utilize the ObIMPDH (●) of the BsuIMPDH (zero) of the BthNK that used embodiment 12 preparations and embodiment 33 preparations or embodiment 34 preparations.
Figure 28 is the figure that interferes the influence of material when utilizing the mizoribine of composition measuring 2 μ g/ml of BsuIMPDH of the BthNK used embodiment 12 preparations and embodiment 33 preparations.About interfering material, be to be to be to be haemoglobin among chyle, (D) among cholerythrin C, (C) among cholerythrin F, (B) in (A).
Figure 29 is the figure of calibration curve that show to utilize the composition measuring mizoribine of the BthNK (●) that used embodiment 12 preparations or the BthNK (zero) of embodiment 13 preparations and the BsuIMPDH that embodiment 33 prepares.
Figure 30 is the figure of calibration curve of composition measuring mizoribine that show to utilize the BsuIMPDH of the BthNK that used embodiment 13 preparations and embodiment 33 preparations.About measurement range, be to be 0~0.5 μ g/ml among 0~7.5 μ g/ml, (B) in (A).
Figure 31 be show to utilize the BthNK that used embodiment 11 preparations and embodiment 33 preparations the composition measuring of BsuIMPDH with the calibration curve (●) of the mizoribine of serum dilution with measure the figure of the calibration curve (zero) of the mizoribine that dilutes with hematoclasis liquid.
Figure 32 utilizes mizoribine assay method of the present invention (X-axis) and the mizoribine of the embodiment 1 described HPLC of utilization of composition of the BsuIMPDH of the BthNK used embodiment 12 preparations and embodiment 33 preparations to measure the graph of a relation of (Y-axis).
Figure 33 is the figure that shows the upright guest's of composition measuring phosphoimidazole who utilizes the BsuIMPDH that has used embodiment 33 preparations calibration curve.
Figure 34 utilizes the mizoribine assay method of the present invention (Y-axis) of composition of the BsuIMPDH of the BthNK used embodiment 12 preparations and embodiment 33 preparations to measure the graph of a relation of (X-axis) with utilizing Journalof chromatography, 432 volumes,, the mizoribine of the method for the use HPLC of 340-345 page or leaf record in 1988.
Embodiment
The present invention relates to comprise the mizoribine of following (1) operation and/or the assay method of Ribavirin.(1) comprises the operation of first reaction, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine and/or the Ribavirin that may contain in the sample carried out phosphorylation mizoribine and/or Ribavirin.
Mizoribine of the present invention or Ribavirin comprise known mizoribine or Ribavirin (CAS numbering 50924-49-7 or 36791-04-5), are not originated, the qualification of formulation, crystal type, additive, trade name etc.
Upright guest of phosphoimidazole of the present invention or phosphoric acid Ribavirin are mizoribine or the Ribavirins that is combined with phosphate.The quantity of phosphate can be 1,2 or 3, and the quantity of preferably phosphoric acid base is 1, the upright guest of promptly preferred single phosphoimidazole or single phosphoric acid Ribavirin.For being not particularly limited by the position of phosphorylation, can be that its sugar moieties is any hydroxyl of ribose part, can enumerate 1 ', 2 ' or 5 ', can also form ring sometimes, situation with the upright guest of single phosphoimidazole or single phosphoric acid Ribavirin is an example, and preferred combination is at 5 '.That is, the upright guest of this list phosphoimidazole or preferred mizoribine 5 ' one phosphoric acid of single phosphoric acid Ribavirin or Ribavirin 5 ' one phosphoric acid.
Have among the above-mentioned physicochemical property<enzyme of 1〉effect character of the present invention can comprising the enzyme that mizoribine and/or Ribavirin carry out phosphorylation.In this instructions, can make first enzyme to the enzyme note that mizoribine and/or Ribavirin carry out phosphorylation (comprising catalysis first reaction) sometimes.Can there be this first enzyme in the operation of assay method of the present invention (1), as this first enzyme, from its physicochemical property aspect,, be not subjected to the restriction of enzyme title, EC numbering or manufacture method etc. so long as can the enzyme that mizoribine and/or Ribavirin carry out phosphorylation be got final product.As this first enzyme, preferably have above-mentioned<1 〉~<4 physicochemical property or the enzyme of any character in the character of this first enzyme of putting down in writing of present specification.Promptly, preferably have above-mentioned<1 〉~<4 physicochemical property among<1 the effect, for having or not other character to have no particular limits, also preferably have above-mentioned<1 〉~<4 physicochemical property or the character of this first enzyme of putting down in writing of present specification among 1 or 2 above character arbitrarily.For first enzyme of the present invention, can also adopt other physicochemical properties, physical property of the character of this first enzyme that present specification puts down in writing etc.
First enzyme of the present invention then is not particularly limited so long as can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, and the enzyme of selecting at least the object that will measure to be carried out phosphorylation gets final product.For example, can enumerate the enzyme that can maybe can carry out phosphorylation to the either party in mizoribine or the Ribavirin to the enzyme that mizoribine and Ribavirin carry out phosphorylation.It is that first enzyme of the present invention comprises: the enzyme that can carry out the phosphorylation of equal extent to mizoribine and Ribavirin; Can carry out phosphorylation to mizoribine and Ribavirin, but can more effectively carry out the enzyme of phosphorylation a certain side in mizoribine or the Ribavirin; Can only optionally carry out the enzyme of phosphorylation to a certain side in mizoribine or the Ribavirin.
First enzyme of the present invention be so long as can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, then without limits to its source, and for example can be from natural biological, but the aspect that obtains enzyme from easy cultivation, easily considers, preferably derives from the enzyme of microorganism.As first enzyme of the present invention that preferably derives from microorganism, can enumerate and for example derive from that bulkholderia cepasea belongs to or enzyme that the Xanthomonas campestris (Xanthomonas) of its nearly edge belongs to etc., preferably derive from the enzyme of Thailand's bulkholderia cepasea, most preferably derive from the enzyme of Thailand bulkholderia cepasea DSM13276 strain.This bacterial strain can be bought by strain library described later, also can use this bacterial strain of buying by other strain libraries equal bacterial strain, use Epidemiol.Infect., the isolated equal bacterial strain of method that 118 volume 137-148 pages or leaves, nineteen ninety-five are put down in writing, after confirming its enzyme that can generate each character, use it for assay method of the present invention with enzyme of the present invention.
In addition, as first enzyme of the present invention, can also use the nucleoside kinase that comprises adenosine kinase, phosphofructokinase-B or phosphatase etc.As first enzyme of the present invention that derives from microorganism, can enumerate be considered to derive from colibacillary guanosine inosine kinase, derive from Methanococcus jannaschii (Methanocaldococcus jannaschii) phosphofructokinase-B, derive from the protein of phosphofructokinase-B of the ancient green-ball bacterium of flicker (Archaeoglobus fulgidus).As first enzyme of the present invention that derives from other microorganisms, can enumerate the adenosine kinase that can carry out phosphorylation that derives from Saccharomyces cerevisiae, mycobacterium to mizoribine and/or Ribavirin.In addition, be difficult to obtain though derive from mammiferous enzyme, and efficient is also not necessarily high, such enzyme also can use, and for example can enumerate the adenosine kinase that derives from the people or derive from the known adenosine kinases of mouse, rat, rabbit etc.
First enzyme of the present invention is so long as can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, unqualified to its amino acid sequence, for example can be disappearance in the amino acid sequence of sequence table sequence numbering 1, replace or added one or more amino acid whose amino acid sequence.This amino acid sequence be preferably the amino acid sequence that contains ordered list sequence numbering 5 and sequence table sequence numbering 6 amino acid sequence sequence (wherein, Xaa in the sequence table sequence numbering 5 and 6 represents amino acid arbitrarily), most preferably be the amino acid sequence of sequence table sequence numbering 1.Example as amino acid whose disappearance, replacement or interpolation in the amino acid sequence of sequence table sequence numbering 1, can enumerate at the N of first enzyme end side and/or C end side and add functional enzyme such as thioredoxin enzyme or the example of the part that constitutes by other amino acid sequence, also preferably make fused protein, can enumerate and make with extra care with the part that can utilize this interpolation or the situation of the partial fusion that is known as mark (tag) of affirmation etc., sometimes, this mark part can be removed, sometimes all or part of that also can residual this part.For example, can add be used for first enzyme outside thalline, about 20 signal peptides of cell pericentral siphon transmission or be used to carry out 5~10 effectively refining His, when adding these, can be linked to be row.In addition, the place such as several proteinase identification amino acid sequences can also be configured between these amino acid sequences.Can similarly lack with the example of above-mentioned interpolation or replace, for example, under the situation of existence by the domain that constitutes with the irrelevant several amino acid of the essential function of first enzyme of the present invention, exist under the situation by two in the amino acid sequence of sequence table sequence numbering 1 breach that constitute with upper amino acid, also can make up these amino acid whose disappearances.In addition, but also appropriate combination disappearance, replace or add.As such amino acid sequence, more preferably contain the enzyme that can carry out phosphorylation of sequence RREFGGCA (G, A or T) NI (A or G) (Y or F) (A, S, T or N) L and DPTG (C, A, V or I) GDA (F, Y, W or H) R (G, A or S) G to mizoribine and/or Ribavirin.In addition, in the amino acid sequence of sequence table sequence numbering 1, the N end side of the amino acid sequence of this sequence table sequence numbering 1 and C end side can contain and promptly be added with amino acid residue or polypeptid residue, add amino acid residue as this, can enumerate signal peptide, TEE sequence, S mark or His mark etc.Under the situation of the amino acid deletions of sequence table sequence numbering 1, for example, can enumerate from N end side or C end side and excise amino acid whose example successively.Having lacked the enzyme of Met of N end or N end in the amino acid sequence of sequence table sequence numbering 1, to be subjected to the enzyme through posttranslational modification of modifications such as acyl group, alkyl etc. also be first enzyme of the present invention.In addition, can also utilize known method, utilize succinic anhydride, PEG etc. that first enzyme is carried out chemical modification, so that change of properties such as the optimal pH of first enzyme, stability are the state that is easy to be utilized.Under the above-mentioned situation, the molecular weight of first enzyme can change sometimes, for example, utilizes pTipQC1 described later, pTip QC2 when the terminal His of interpolation of N mark, and molecular weight is big about 1000.Secondary structure, three aggregated(particle) structures and four aggregated(particle) structures to the amino acid sequence of first enzyme of the present invention are not particularly limited.
The base sequence of first enzyme of the present invention is so long as the base sequence of first enzyme of the present invention of encoding, then have no particular limits, for example in the situation of the base sequence of the amino acid sequence of code sequence tabulation sequence numbering 1, can be coding and the base sequence of the impartial in fact amino acid sequence of sequence table sequence numbering 1, for example can be the amino acid sequence of variation to have taken place (as disappearance with the irrelevant a part of amino acid of the catalytic action of above-mentioned first reaction among the amino acid sequence of sequence table sequence numbering 1, replace or added the equipollent of one or more amino acid whose original acid sequence) base sequence of encoding.The base sequence that especially preferably contains the amino acid sequence separately of code sequence tabulation sequence numbering 5 and sequence table sequence numbering 6.As the base sequence of the amino acid sequence of code sequence tabulation sequence numbering 1, can enumerate the base sequence of sequence table sequence numbering 2 or the base sequence of sequence table sequence numbering 2 carried out the base sequence that changes in conjunction with hosts' such as Escherichia coli, actinomyces codon usage frequency.
When preparing the base sequence of coding first enzyme, normally used known genetic manipulation means be can utilize, the alternative method of site specific, the whole bag of tricks such as method that usefulness artificial variation base replaces the particular bases fragment of genes of interest for example can be enumerated.
In this instructions, first enzyme note that will be made of the amino acid sequence of sequence table sequence numbering 1 is sometimes made BthNK.
As long as can measure mizoribine and/or Ribavirin, amount to first enzyme of use in the operation (1) of assay method of the present invention is not particularly limited, can carry out the purity of device, first enzyme of purpose degree, the use of phosphorylation and/or economic situation etc. according to the amount of mizoribine that sample contained and/or Ribavirin, to mizoribine and/or Ribavirin, determine an amount that can access desired result.Also having used in the operation of assay method of the present invention (1) under the situation of phosphodonor, metallic ion, also is identical for its kind with amount.
For example, condition be the amount of the mizoribine that contains of raw material, sample and/or Ribavirin be below the 1mM and will situation with its whole phosphorylations under, the following of amount of first enzyme that uses in the operation of assay method of the present invention (1) is limited to more than the 0.01U/ml, be preferably more than the 0.05U/ml, more preferably more than the 0.1U/ml, the upper limit is not particularly limited, but is generally below the 5U/ml, be preferably below the 2U/ml, more preferably below the 1U/ml.
The operation of assay method of the present invention (1) is preferably carried out in the presence of phosphodonor at least, and this phosphodonor also can be CTP, GTP, TTP or polyphosphoric acid etc., but preferred especially ATP.With ATP is example, more than the following 0.1mM of being limited to of the amount of the phosphodonor of using under the above-mentioned condition, is preferably more than the 1mM, more preferably more than the 5mM, the upper limit is not particularly limited, but is preferably below the 500mM, more preferably below the 200mM, be preferably below the 50mM especially.
The operation of assay method of the present invention (1) is preferably carried out in the presence of metallic ion at least, and described metallic ion can be any one above metallic ion among magnesium ion, cobalt ions, nickel ion or the manganese ion, but special preferably magnesium ion.With the magnesium ion is example, and the amount of the metallic ion that uses under the above-mentioned condition following is limited to more than 0.1 equivalent of concentration of phosphodonor, is preferably more than 0.5 equivalent, more preferably more than 1 equivalent, on be limited to below 10 equivalents, be preferably below 5 equivalents, more preferably below 3 equivalents.Most preferred concentration is 2 equivalents of phosphodonor.
Assay method of the present invention precision is under various conditions implemented to measure exactly, and this point can obtain the support of instructions embodiment of the present invention.Sample of the present invention is had no particular limits, can enumerate the Biosample, research sample and these the extract etc. that contain whole blood, blood plasma, serum, blood cell, marrow liquid, lymph liquid, urine etc., these samples are preferably inferred the sample that contains mizoribine and/or Ribavirin, are more preferably to infer the sample that contains a certain side in mizoribine or the Ribavirin.Sample is under the situation of Biosample, also preferably choose the target internal organs (for example white blood cell, liver cell, virus infected cell etc.) of mizoribine and/or Ribavirin and make sample, sample is under the leukocytic situation, also preferably chooses T cell, B cell, lymph corpuscle etc. and makes sample.Gathering supposition contains under the Biosample situation of mizoribine and/or Ribavirin, acquisition method can be a known method, gather under the situation of whole blood, to whether using separating agent, antiplasmin agent etc. without limits, to whether using anticoagulant, glycolytic inhibitors such as EDTA, sodium fluoride, sodium citrate, liquaemin also without limits.As other samples, can enumerate for example seawater, natural water, fruit juice, beverage, waste liquid etc.
Judge in the time of whether may containing mizoribine and/or Ribavirin in the sample, be included in and implement to judge the situation that whether may contain mizoribine and/or Ribavirin in the sample before the assay method of the present invention, for example can wait to judge whether and to contain, can also judge whether possible by above-mentioned prior aries such as HPLC, LC/MS/MS by medication experience, diet experience.In addition, can also judge in the sample whether may contain by implementing assay method of the present invention.Equally, judge also to utilize prior art and method described later in the time of whether may containing upright guest of phosphoimidazole and/or phosphoric acid Ribavirin in the sample.Usually can by medication through always judging in the sample whether may contain mizoribine and/or Ribavirin.
Among the present invention, can there be mizoribine simultaneously in the sample, Ribavirin, upright guest of phosphoimidazole and phosphoric acid Ribavirin, to there being mizoribine simultaneously, Ribavirin, under the situation that the mizoribine in the sample of upright guest of phosphoimidazole and phosphoric acid Ribavirin or the amount of Ribavirin are measured, can be suitably with the method for the amount of assay method of the present invention and prior art or upright guest of mensuration phosphoimidazole described later and/or phosphoric acid Ribavirin and/or the above-mentioned HPLC that utilizes, the method of the amount of LC/MS/MS mensuration mizoribine or Ribavirin makes up to be implemented, and calculates mizoribine by deducting these numerical value, the amount of Ribavirin.Usually preferably the sample of individualism mizoribine or Ribavirin is measured.
The present invention also contains the mizoribine of following (2) operation and/or the assay method of Ribavirin.(2) suppress second operation of reacting, wherein, can catalysis second reaction second enzyme in the presence of, when carrying out second reaction, upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin that above-mentioned first reaction is generated contact with second enzyme, make second reaction be subjected to the inhibition of the degree corresponding with the amount of upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin, described second reaction is and the different reaction of above-mentioned first reaction, is following<a 〉~<c in any one reaction:
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses;
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses;
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin.
In this instructions, can this second enzyme note of reacting of catalysis make second enzyme sometimes.Can there be this second enzyme in the operation of assay method of the present invention (2),,, not numbered by enzyme title, EC and/or the restriction of manufacture method etc. so long as the enzyme of can catalysis above-mentioned second reaction get final product from its physicochemical property aspect.This second enzyme can be can catalysis not to be subjected to mizoribine and Ribavirin the two suppresses, but is subjected to the enzyme of the reaction of upright guest of phosphoimidazole that above-mentioned first reaction generates and the two inhibition of phosphoric acid Ribavirin.In addition, this second enzyme can also be catalysis not suppressed by mizoribine, but the enzyme of the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses.Moreover this second enzyme can also be catalysis not suppressed by Ribavirin, but the enzyme of the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses.As this second enzyme, preferably have above-mentioned<i 〉~<vi physicochemical property or the enzyme of any character in the character of this second enzyme of putting down in writing of present specification, above-mentioned<i 〉~<vi physicochemical property among preferably have<v inhibiting effect, have no particular limits for the character that has or not other, also preferably have above-mentioned<i 〉~<vi the character of physicochemical property, this second enzyme that present specification is put down in writing among any character more than 1 or 2.The enzyme of reaction of inhibition that more preferably can catalysis be subjected to the amount degree of correspondence of the upright guest of phosphoimidazole that generates with first reaction and phosphoric acid Ribavirin, most preferably inosine 5 ' monophosphate dehydrogenase.Inosine 5 ' monophosphate dehydrogenase is expressed as EC 1.1.1.205 sometimes.Adopt under the situation of inosine 5 ' monophosphate dehydrogenase as second enzyme of the present invention, to the source without limits, for example can derive from natural biological, in addition, derive under the situation of microorganism, for example can be to derive from the people, Escherichia coli, Candida albicans (Candida albicans), Tritrichomonas foetus (Tritrichomonasfoetus), Borrelia burgdoyferi (Borrelia burgdorferi), rat, rabbit, the known inosine 5 ' monophosphate dehydrogenase of bacillus cereus (Bacillus cereus) etc. also can be these isoenzymes (S.F.Carr etc., J.Biol.Chem., 268 volumes, 27286 pages, 1993; H.J.Gilbert etc., Biochem.J., 183 the volume, 481 pages, 1979; G.A.Koehler etc., J.Bacteriol., 179 the volume, 2331 pages, 1997; F.G.Whitby etc., 36 the volume, 10666 pages, 1997; X.Zhou etc., J.Biol.Chem., 272 volumes, 21977 pages, 1997 etc.).Typical case as the natural microbial that forms the inosine 5 ' monophosphate dehydrogenase that can be used as second enzyme, can enumerate the microorganism that belongs to bacillus, bacillus marinus genus, more preferably hay bacillus or Oceanobacillus iheyensis, most preferably hay bacillus ATCC23857 strain or Oceanobacillusiheyensis DSM14731 strain.In addition, also can use the equal bacterial strain of this bacterial strain of buying by other strain libraries or use with said method, after affirmation can generate the enzyme of each character with enzyme of the present invention, use it for assay method of the present invention from the equal bacterial strain that nature separates.
Second enzyme of the present invention is so long as the enzyme of can catalysis above-mentioned second reaction, to its amino acid sequence without limits, for example under the situation for the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9, it can be disappearance, replace or added one or more amino acid whose amino acid sequence, but this amino acid sequence preferably contains the amino acid sequence of ordered list sequence numbering 11 and the amino acid sequence of sequence table sequence numbering 12, the sequence that more preferably contains ordered list sequence numbering 23, the sequence that further preferably contains ordered list sequence numbering 24 is preferably the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9 especially.Aspect plural amino acid deletions, replacement or interpolation in sequence table sequence numbering 7 of the present invention or sequence table sequence numbering 9, posttranslational modification, the chemical modification, the configuration aspects of its final change of molecular weight aspect, amino acid sequence, all the situation with above-mentioned first enzyme is identical.Second enzyme of the present invention is under the situation of for example amino acid sequence of sequence table sequence numbering 7, as the example that has replaced one or more amino acid whose amino acid sequence, can enumerate the combination in any of V432I (432 V of expression sequence table sequence numbering 7 is replaced to I, down together), K449I, K345N, D29N, S33Y, T158S, T270A, V387E and L367I and these replacements.This makes up arbitrarily for example can enumerate [V432I, K449I] (V of 432 of expression sequence table sequence numbering 7 is replaced to I and while 449 K is replaced to I, down together), [D29N, S33Y, T158S, T270A, V387E].
The base sequence of second enzyme of the present invention is so long as the base sequence of second enzyme of the present invention of encoding, then have no particular limits, can enumerate the amino acid sequence of code sequence tabulation sequence numbering 7 or sequence table sequence numbering 9 base sequence, contain the variation identical, the base sequence of change with the situation of above-mentioned first enzyme.
When preparing the base sequence of coding second enzyme of the present invention, can utilize the genetic manipulation means identical with the situation of above-mentioned first enzyme.
In this instructions, second enzyme note that will be made of the amino acid sequence of sequence table sequence numbering 7 is sometimes made BsuIMPDH, and second enzyme that will be made of the amino acid sequence of sequence table sequence numbering 9 note is made ObIMPDH.
As long as can measure mizoribine and/or Ribavirin, amount to second enzyme of use in the operation (2) of assay method of the present invention is not particularly limited, can carry out the purity of device, second enzyme of purpose degree, the use of phosphorylation and/or economic situation etc. according to the amount of mizoribine that sample contained and/or Ribavirin, to mizoribine and/or Ribavirin, determine an amount that can access desired result.Use in the operation of assay method of the present invention (2) under the situation of substrate corresponding to employed second enzyme, coenzyme, perhaps use under the situation of inosine 5 ' one phosphoric acid, NAD (P) class, for its kind with to measure also be identical.
For example, the amount of mizoribine that raw material, sample contained and/or Ribavirin be below the 1mM and will be with it whole phosphorylations, use under the situation of inosine 5 ' monophosphate dehydrogenase as second enzyme, the following of amount of second enzyme that uses in the operation of assay method of the present invention (2) is limited to more than the 0.01U/ml, be preferably more than the 0.05U/ml, more preferably more than the 0.1U/ml, the upper limit is not particularly limited, but be generally below the 1U/ml, be preferably below the 0.5U/ml, more preferably below the 0.3U/ml.
The operation of assay method of the present invention (2) is preferably carried out in the presence of inosine 5 ' one phosphoric acid at least, and this inosine 5 ' one phosphoric acid does not limit the kind of salt and has or not water of crystallization.More than the following 1mM of being limited to of the amount of inosine 5 ' one phosphoric acid that uses under the above-mentioned condition, be preferably more than the 3mM, more preferably more than the 5mM, the upper limit is not particularly limited, but be preferably below the 100mM, more preferably below the 50mM, be preferably below the 30mM especially.
The operation of assay method of the present invention (2) is preferably carried out in the presence of NAD (P) class at least, can enumerate NAD (P), deacylated tRNA amine NAD (P) (NAAD (P)), sulfo-NAD (P), niacinamide guanine dinucleotide (phosphoric acid), niacinamide FAD (phosphoric acid) etc. in NAD (P) class, can be wherein more than any one, preferred especially NAD.For example under the situation of NAD, more than the following 0.1mM of being limited to of the amount of the NAD that uses under the above-mentioned condition (P) class, be preferably more than the 0.5mM, more preferably more than the 1mM, the upper limit is not particularly limited, but is preferably below the 50mM, more preferably below the 10mM, be preferably below the 7mM especially.
Assay method of the present invention is to comprise the mizoribine of following (3) operation and/or the assay method of Ribavirin.(3) detect the operation that second reaction is suppressed degree.
The operation of detection method of the present invention (3) can be to detect the operation of inhibition degree by qualitative reaction, the detection of preferred inhibition degree is the operation that detects the reaction velocity of second enzyme that can catalysis second reaction, the detection that more preferably suppresses degree is the operation that detects the variable quantity of NAD (P) class, and the detection that most preferably suppresses degree is to detect the variable quantity of NAD (P) class and quantitative operation.
It is as follows to carry out quantitative assay method.The sample that may contain mizoribine and/or Ribavirin is handled with operation (1)~(3) separately respectively with the sample of mizoribine that contains concentration known and/or Ribavirin, at operation (3) detection variable quantity separately.To do thus the concentration of mizoribine in the sample that may contain mizoribine and/or Ribavirin and/or Ribavirin to be carried out quantitatively to the detected variable quantity of the sample of the mizoribine that contains concentration known and/or Ribavirin to the detected variable quantity of the sample that may contain mizoribine and/or Ribavirin with by operation (3) by operation (3) with relatively.
In the method for the variable quantity of detection NAD (P) class, detect the variable quantity of this determination object with known method.Generally can enumerate operative installations detects absorption spectrum, the photon absorbing intensity of this determination object that the variation follow this determination object takes place by optical means the method for variation, also can be the method that detects the variation of absorption spectrum that the redox of following NAD (P) class produces, the photon absorbing intensity under the specific wavelength.In addition, also can detect the change in fluorescence of NAD (P) H class.Based on improving sensitivity or, can also making wherein to have DI (P) (diaphorase, Diaphorase) Huo Jia Za (formazan) pigment by purposes such as visual detection.In addition, in order further to improve sensitivity etc., can also adopt NAD (P) class circular response.When measuring the potential difference (PD) due to the redox of NAD (P) class, use electrode.The enzyme that uses can be fixed in carbon paste etc. and make electrode, be used for the mensuration of mizoribine and/or Ribavirin.
When implementing assay method of the present invention, can perhaps implement, preferably implement in liquid phase in enforcements such as liquid phase, gas phase or solid phases at critical surface separately.Liquid phase comprises that water, organic solvent equate, implements with water under the situation of assay method of the present invention, and water for example can be enumerated water, aqueous solution, contain the aqueous medium of suitable organic solvent, and preferably uses suitable pH buffering agent.Use under the situation of pH buffering agent, as long as can keep purpose pH and can measure mizoribine and Ribavirin in the sample, then its kind is had no particular limits, can enumerate Good ' s pH damping fluid, Tris/HCl damping fluid, kaliumphosphate buffer, acetate/NaOH damping fluid, citric acid/NaOH damping fluid.As long as can measure mizoribine and/or Ribavirin in the sample, pH when implementing assay method of the present invention has no particular limits, in the operation (1), the lower limit of pH can be enumerated to more than the pH4, be preferably more than the pH4.8, more preferably more than the pH5.2, the upper limit can be enumerated to below the pH8.5, be preferably below the pH8, more preferably below the pH7.5.In operation (2) and (3), lower limit can be enumerated to more than the pH5.5, is preferably more than the pH6, and more preferably more than the pH7, the upper limit can be enumerated to below the pH10.5, is preferably below the pH9.5, more preferably below the pH8.5.As long as can keep purpose pH and can measure mizoribine and Ribavirin, concentration to the pH buffering agent has no particular limits, lower limit can be enumerated to more than the 3mM, be preferably more than the 5mM, more preferably more than the 10mM, the upper limit can be enumerated to below the 500mM, is preferably below the 200mM, more preferably below the 100mM.
As other preferred liquid phase of assay method of the present invention, can enumerate for example sol-gel.In order to make sol-gel, can utilize for example polysaccharide such as agar.Distinguish under the situation of sol-gel and emulsion, also can be made into emulsion and implement to measure.In order to make emulsion, for example can utilize organic solvent etc., when utilizing parents matchmaker's property material, also can be made into micella and implement to measure.Under any situation, if use the pH buffering agent, its situation is same as described above.
(1) in the assay method of the present invention~(3) each operation can be implemented in different reactive tank (phase) respectively, but preferably implements in same reactive tank (phase).In addition, each operation of (1)~(3) can be interrupted enforcement, but preferred enforcement continuously.In addition, each operation of (1)~(3) can be undertaken by the order of (1), (2), (3); Also can carry out (1) and (2) simultaneously, carry out then (3); Can also carry out earlier (1), carry out simultaneously then (2) and (3); Can also carry out (1), (2), (3) simultaneously; Deng, according to definite orders that can access desired result such as device of purpose, sample, use.In addition, when the operation (1) of not implementing assay method of the present invention is just implemented the method for operation (2) and operation (3), can measure the amount of upright guest of phosphoimidazole and phosphoric acid Ribavirin.
As long as can measure mizoribine and/or Ribavirin in the sample, reaction time of operation of the present invention (1)~(3) is had no particular limits, following being limited to more than 15 seconds separately was preferably more than 1 minute, more preferably more than 3 minutes.The upper limit is not provided with especially, but is preferably below 30 minutes, more preferably below 15 minutes, be preferably especially below 10 minutes.(1)~(3) reaction time of each operation can be unequal.
The temperature of implementing assay method of the present invention is had no particular limits, so long as can measure mizoribine in the sample and/or the temperature of Ribavirin gets final product, preferably in the scope of the operative temperature of employed enzyme, and, be limited to more than 15 ℃ down, be preferably more than 20 ℃, more preferably more than 25 ℃, on be limited to below 70 ℃, be preferably below 50 ℃, more preferably below 40 ℃.(1)~(3) temperature of each operation can be unequal.
As of the present invention and the mizoribine of the operation that comprises above-mentioned (1)~(3) and/or other different assay methods of assay method of Ribavirin, can be the assay method that comprises following (a)~(c) each operation.(a) comprise the operation of reacting I, in described reaction I, in the presence of above-mentioned first enzyme and ATP, make the mizoribine and/or the Ribavirin that may contain in the sample become the upright guest of phosphoimidazole and/or phosphoric acid Ribavirin and ADP; (b) in the presence of the enzyme B of catalysis reaction II different or plural reaction II or plural enzyme B with above-mentioned reaction I, the operation that the amount of the ADP that the corresponding above-mentioned reaction I of the determination object that reaction II is produced produces changes; (c) detect the variable quantity of this determination object, measure the operation of the amount of the mizoribine that may contain in the sample and/or Ribavirin.
To the source of mizoribine in other assay methods of mizoribine of the present invention and/or Ribavirin and/or Ribavirin without limits, but preferred above-mentioned sample.Implement condition, the reactive tank (mutually) of condition, time and the temperature of the phase of other assay methods of mizoribine of the present invention and/or Ribavirin, the pH buffering agent when implementing with liquid phase condition, implement operation sequential scheduling same as described above.
The amount of first enzyme that in the operation (a) of other assay methods of mizoribine of the present invention and/or Ribavirin, uses, use in conditions such as used kind and amount under the situation of phosphodonor and metallic ion etc. and the above-mentioned operation (1) identical.That is, the operation of other assay methods of mizoribine of the present invention and/or Ribavirin (a) can be implemented with the condition identical with operation (1) in the assay method of the present invention.
The operation of other assay methods of mizoribine of the present invention and/or Ribavirin (b) is in the presence of the enzyme B of catalysis reaction II different with above-mentioned reaction I or plural reaction II or plural enzyme B, the operation that the amount of the ADP that the corresponding above-mentioned reaction I of the determination object that reaction II is produced produces changes.
Example as the operation (b) of other assay methods of mizoribine of the present invention and/or Ribavirin, can enumerate following operation: (b) operation comprises (b-1) reaction and (b-2) reaction, (b-1) in the reaction, in the presence of ADP dependence hexokinase, make glucose and ADP become G-6-P and AMP; (b-2) in the reaction, in the presence of glucose-6-phosphate dehydrogenase (G6PD), the G-6-P that NAD (P) class and above-mentioned reaction are generated becomes NAD (P) H class and gluconolactone-6-phosphoric acid; The amount of the ADP that the corresponding above-mentioned reaction I of NAD (P) H class generates increases (this instructions remember sometimes make operation (b (α))), (c) detects the recruitment of NAD (P) H class in the operation.
As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then the ADP dependence hexokinase in the operation (b (α)) of other assay methods of mizoribine of the present invention and/or Ribavirin and the source or the origin of glucose-6-phosphate dehydrogenase (G6PD) are not particularly limited.As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then the ADP dependence hexokinase in the operation (b (α)) and the concentration of glucose-6-phosphate dehydrogenase (G6PD) are had no particular limits, for example, under be limited to more than the 0.1U/ml, be preferably more than the 1U/ml, more preferably more than the 2U/ml, do not have special capping, be preferably below the 20U/ml, more preferably below the 10U/ml.Can exist ADP dependence hexokinase for the desired substrate of reaction, metallic ion in the operation (b (α)).As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then kind and the concentration to substrate, metallic ion has no particular limits, but that this kind and concentration can be because of the source of employed ADP dependence hexokinase or origins is different and different.Make the example of substrate as glucose, for example concentration is more than the 0.01mM, is preferably more than the 0.1mM, more preferably more than the 1mM, does not have special capping, is preferably below the 500mM, more preferably below the 100mM.Make the example of metallic ion as magnesium chloride, for example concentration is more than the 0.1mM, is preferably more than the 1mM, more preferably more than the 5mM, does not have special capping, is preferably below the 100mM, more preferably below the 10mM.Can have NAD (P) class in the operation (b (α)), it is the coenzyme of glucose-6-phosphate dehydrogenase (G6PD).As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then kind, the concentration to NAD (P) class has no particular limits, but that this kind and concentration can be because of the source of employed glucose-6-phosphate dehydrogenase (G6PD) or origins is different and different.Make the example of coenzyme as NAD (P), for example concentration is more than the 0.1mM, is preferably more than the 1mM, more preferably more than the 3mM, does not have special capping, is preferably below the 50mM, more preferably below the 10mM.The kind of NAD (P) class is same as described above.
Other examples as the operation (b) of other assay methods of mizoribine of the present invention and/or Ribavirin, can enumerate following operation: (b) comprise (b-1) reaction and (b-2) reaction in the operation, (b-1) in the reaction, in the presence of pyruvate kinase, make ADP and PEP become pyruvic acid and ATP; (b-2) in the reaction, in the presence of lactic dehydrogenase, the pyruvic acid that NAD (P) H class and above-mentioned reaction are generated becomes NAD (P) class and lactic acid; The amount of the ADP that corresponding above-mentioned first reaction of NAD (P) H class generates reduces (this instructions remember sometimes make operation (b (β))), (c) detects the reduction of NAD (P) H class in the operation.
As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then the pyruvate kinase in the operation (b (β)) of other assay methods of mizoribine of the present invention and/or Ribavirin and the source or the origin of lactic dehydrogenase are not particularly limited.As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then the pyruvate kinase in the operation (b (β)) and the concentration of lactic dehydrogenase are had no particular limits, for example, under be limited to more than the 1U/ml, be preferably more than the 5U/ml, more preferably more than the 10U/ml, do not have special capping, be preferably below the 100U/ml, more preferably below the 50U/ml.Can exist pyruvate kinase for the desired substrate of reaction, metallic ion in the operation (b (β)).As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then kind and the concentration to substrate, metallic ion has no particular limits, but that this kind and concentration can be because of the source of employed pyruvate kinase or origins is different and different.Make the example of substrate as PEP, for example concentration is more than the 0.1mM, is preferably more than the 0.5mM, more preferably more than the 1mM, does not have special capping, is preferably below the 50mM, more preferably below the 10mM.Make the example of metallic ion as magnesium chloride, for example concentration is more than the 0.1mM, is preferably more than the 0.5mM, more preferably more than the 1mM, does not have special capping, is preferably below the 50mM, more preferably below the 10mM.Can have NAD (P) H class in the operation (b (β)), it is the coenzyme of lactic dehydrogenase.As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then kind, the concentration to NAD (P) H class has no particular limits, but this kind and concentration can be because of the source of employed lactic dehydrogenase or origin different and different, also the performance because of the optical length of employed cuvette, tintmeter is different.Cuvette with optical length 1cm is an example, and concentration is more than the 0.01mM, be preferably more than the 0.03mM, more preferably more than the 0.07mM, on be limited to below the 0.2mM, be preferably below the 0.17mM, more preferably below the 0.15mM.
Another other examples as the operation (b) of other assay methods of mizoribine of the present invention and/or Ribavirin, can enumerate following operation: (b) comprise (b-1) reaction and (b-2) reaction in the operation, (b-1) in the reaction, in the presence of pyruvate kinase, make ADP and PEP become pyruvic acid and ATP; (b-2) in the reaction, in the presence of pyruvate oxidase, the pyruvic acid that oxygen, phosphoric acid and above-mentioned reaction are generated becomes hydrogen peroxide and acetyl phosphate; The amount of the ADP that corresponding above-mentioned first reaction of hydrogen peroxide generates increases (this instructions is called operation (b (γ)) sometimes), (c) detects the amount of hydrogen peroxide in the operation with hydrogen peroxide indicator etc.
As long as can implement other assay methods of mizoribine of the present invention and/or Ribavirin, then the pyruvate kinase in the operation (b (γ)) of other assay methods of mizoribine of the present invention and/or Ribavirin and the source or the origin of pyruvate oxidase are not particularly limited.The pyruvate kinase in the operation (b (γ)) and the concentration of pyruvate oxidase are identical with the situation of above-mentioned pyruvate kinase and lactic dehydrogenase.Pyruvate kinase is same as described above for the kind and the concentration of the desired substrate of reaction, metallic ion in the operation (b (γ)).When using peroxidase, hydrogen peroxide indicator in the operation (b (γ)) can also use phenol or TOOS, N such as 4-amino-antipyrine, 3-methyl-2-[4-morpholinodithio quinoline ketone hydrazone isochrome idol, chlorophenol, the chromogen such as phenyl amines such as accelerine and their derivant or the chromogen of leuco form etc.
In the method that the variable quantity to this determination object in the operation of other assay methods of mizoribine of the present invention and/or Ribavirin (c) detects, the method for the variable quantity of detection NAD (P) class is same as described above.Use under the situation of hydrogen peroxide indicator, can detect the variation of following this hydrogen peroxide indicator and the variation of the photon absorbing intensity of the absorption spectrum that takes place, specific wavelength.In addition, detect under the situation of the oxygen that consumes, utilize oxygen electrode.
Detection method in the operation of other assay methods of mizoribine of the present invention and/or Ribavirin (c) can be identical with the operation (3) of the assay method of the invention described above.
The phosphorylation method of mizoribine of the present invention and/or Ribavirin is to use first enzyme of the present invention to make mizoribine and/or Ribavirin become the method for upright guest of phosphoimidazole and/or phosphoric acid Ribavirin.
In addition, the method that the phosphorylation method of mizoribine of the present invention and/or Ribavirin is for example preferably following: use first enzyme of the present invention, in the presence of phosphodonor, make mizoribine and/or Ribavirin become upright guest of phosphoimidazole and/or phosphoric acid Ribavirin at least; Any one above metallic ion, mizoribine and/or Ribavirin, phosphodonor and first enzyme of the present invention contacts among preferably making magnesium ion, cobalt ions, nickel ion or manganese ion at least.Order for contact is not particularly limited.
The mizoribine in the phosphorylation method of the present invention and/or the source of Ribavirin are same as described above.The conditions of the condition of the pH buffering agent under the situation of implementing the phase of phosphorylation method of the present invention, implementing with liquid phase etc., time, temperature etc. are same as described above.In addition, conditions such as the amount of first enzyme that uses in this method, the kind of using these materials under the situation of phosphodonor, metallic ion and amount etc. are same as described above.That is, phosphorylation method of the present invention can be implemented with the condition identical with operation (1) in the assay method of the present invention.
The phosphoimidazole that makes mizoribine and/or Ribavirin become upright guest of phosphoimidazole and/or phosphoric acid Ribavirin of the present invention stands in the manufacture method of guest and/or phosphoric acid Ribavirin, the phosphorylation method of mizoribine and/or Ribavirin by the invention described above is made upright guest of phosphoimidazole and/or phosphoric acid Ribavirin by mizoribine and/or Ribavirin.Upright guest of the phosphoimidazole of manufacturing and/or phosphoric acid Ribavirin can maintenance mix the state that exists with raw material, other manufacturing things etc., but preferably make it substantially not comprise impurity, make for example various purity more than 50%, more than 70%, more than 95% usually according to situations such as purpose, purposes.Can make with extra care upright guest of phosphoimidazole and/or phosphoric acid Ribavirin with known process for purification, for example can enumerate the extraction of using solvent etc., use the chromatography of silica gel, aluminium oxide and/or zeyssatite etc. and/or utilize the process for purification of recrystallization etc.
First enzyme of the present invention and/or second enzyme can be made mizoribine and/or Ribavirin mensuration composition.Mizoribine of the present invention and/or Ribavirin are measured can be used for the assay method of above-mentioned mizoribine of the present invention and/or Ribavirin and other assay methods of mizoribine of the present invention and/or Ribavirin with composition.In addition, also can be used for mizoribine and/or Ribavirin make upright guest of phosphoimidazole and/or phosphoric acid Ribavirin method, mizoribine and/or Ribavirin are made the upright guest of phosphoimidazole of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin and/or the manufacture method of phosphoric acid Ribavirin.
Composition of the present invention can be enumerated the composition that contains following (A1)~(A4-3) composition.Said composition can be any one in the composition of mizoribine mensuration composition, Ribavirin mensuration composition, mizoribine and Ribavirin mensuration.
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to mizoribine and/or Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, its can catalysis second reaction, described second reaction is and the different reaction of above-mentioned first reaction, is following<a 〉~<c in any one reaction:
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses,
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses,
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
The composition that comprises above-mentioned (A1)~(A3) and following containing (A4-1)~(A4-3) composition in the composition of the present invention.Said composition can be that the upright guest of phosphoimidazole measures to measure with composition, phosphoimidazole with composition, phosphoric acid Ribavirin and founds guest and phosphoric acid Ribavirin mensuration composition.
(A4-1) second enzyme, catalysis and the different reaction of above-mentioned first reaction are subjected to the inhibition of phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
In addition, any one above composition adds before being about to mensuration among the preferred following 3 kinds of compositions of composition of the present invention, to make composition of the present invention.That is, when making the composition of the present invention with whole compositions required when measuring, preferably following at least 3 kinds of compositions or a kind of arbitrarily or 2 kinds of compositions separate with composition before measuring.With among 3 kinds of following compositions any one, two or three composition merges to before be about to measuring under the situation in the composition, the order of merging is arbitrarily, can merge one by one, also can merge 2 or 3 simultaneously.
(A4-1) second enzyme,
(A4-2) inosine 5 ' one phosphoric acid and
(A4-3) NAD (P) class.
It is to contain following (A) and composition (B) that mizoribine of the present invention and/or Ribavirin are measured with composition.
(A) the 1st reagent, it contains following (A1)~(A4) at least:
(A1) first enzyme of the present invention of effective dose,
(A2) phosphodonor,
(A3) metallic ion,
(A4) the above composition of any one in second enzyme of the present invention of effective dose, inosine 5 ' one phosphoric acid and NAD (P) class;
(B) the 2nd reagent, it contains the composition that do not contain in the 1st reagent among second enzyme, inosine 5 ' one phosphoric acid and NAD (P) class that upright guest of phosphoimidazole and/or phosphoric acid Ribavirin suppress of being subjected to of effective dose at least.
Mizoribine of the present invention and/or Ribavirin are measured with composition and are preferably contained following (A) and (B).
(A) the 1st reagent, it contains following (A1)~(A4) at least:
(A1) first enzyme of the present invention of effective dose,
(A2) phosphodonor,
(A3) metallic ion,
(A4) second enzyme of the present invention and NAD (P) class;
(B) the 2nd reagent, it contains inosine 5 ' one phosphoric acid of effective dose at least.
The mizoribine of the invention described above and/or Ribavirin are measured with composition (A) and (B) are also preferably contained suitable pH buffering agent.
Mizoribine of the present invention and/or Ribavirin are measured can also contain following (C) with composition.(C) standard reagent, it contains the mizoribine and/or the Ribavirin of known quantity at least.
The kind that mizoribine of the present invention and/or Ribavirin are measured with first enzyme that composition contained, of the present invention, second enzyme of the present invention, phosphodonor, metallic ion, inosine 5 ' one phosphoric acid and NAD (P) class is that said composition becomes mizoribine and/or Ribavirin mensuration uses the required effective addition of composition and the condition of pH buffering agent etc. identical with above-mentioned assay method of the present invention with effective dose.
Standard reagent of the present invention can be to contain the mizoribine of known quantity and/or the reagent of Ribavirin at least, is preferably and contains the pH buffering agent; Antiseptic such as sodium azide, microbiotic; The reagent of stabilizing agents such as sugar.Contain under the situation of pH buffering agent, conditions such as its kind, concentration etc. are identical with the situation of the operation (1) of assay method of the present invention.Contain under the situation of sodium azide, microbiotic etc., then unqualified as long as have antiseptic effect to its kind, concentration, for example be example with the sodium azide, be limited to more than 0.005% down, be preferably more than 0.01%, more preferably more than 0.03%; On be limited to below 1%, be preferably below 0.5%, more preferably below 0.1%.For example be example, be limited to more than the 5 μ g/ml down, be preferably more than the 10 μ g/ml, more preferably more than the 30 μ g/ml with the antibiotics; On be limited to below the 100 μ g/ml, be preferably below the 75 μ g/ml, more preferably below the 60 μ g/ml.Contain under the situation of stabilizing agent, conditions such as its kind, concentration etc. are identical with the stabilizing agent of above-mentioned first enzyme.Calibration steps can be selected the linear regression of single-point detection method, multiple spot detection method (broken line or batten (ス プ ラ イ Application)) or multiple spot detection etc.
Be not particularly limited for this known quantity, as long as select accurately to measure the mizoribine in the sample and/or the amount of Ribavirin.In addition, use under the situation of plural standard reagent, the known quantity of standard reagent is also identical.When for example being example, be limited to more than the 0.00 μ g/ml down, be preferably more than the 0.005 μ g/ml, more preferably more than the 0.01 μ g/ml with the mizoribine; On be limited to below the 20 μ g/ml, be preferably below the 10 μ g/ml, more preferably below the 8 μ g/ml.
Mizoribine of the present invention and/or Ribavirin are measured with composition and standard reagent and can be provided with the form of the dry product (obtaining by modes such as heat drying and/or air-dry and/or drying under reduced pressure) of the freeze drying product of the frozen material of liquid state product, liquid product, liquid state product or liquid state product.The frozen material of preferred liquid product, more preferably the freeze drying product of liquid product, most preferably liquid state product.As other modes, the frozen material of also preferred liquid product sometimes.As other other modes, the freeze drying product of preferred sometimes liquid state product.It can be the composition of single reagent that mizoribine of the present invention and/or Ribavirin are measured with composition, but usually preferred above-mentioned being divided into like that more than two reagent.In addition, based on the purposes such as quality that improve reagent, can mix salt such as NaCl, KCl therein; Surfactant such as TX-100, Tween20; And/or antiseptics such as sodium azide, microbiotic.In addition, for example be used for the capillary column of POC or situation about using as enzyme sensor under, preferably the concentration of each composition is greater than at ordinary times concentration, for example preferably carries out immobilization or inhale being imprinted on paper or film or making gel-sol shape composition and use.Under the situation of salt-mixture, kind, concentration to salt are unqualified, common scope is 5~200mM, under the situation of mixed surfactant, kind, concentration to surfactant are unqualified, be generally 0.001%~2%, mix under the situation of antiseptic, identical with the situation of above-mentioned standard reagent.
Manufacture method to first enzyme of the present invention describes below.
For first enzyme of the present invention, can seek the natural biological that (screening) forms this first enzyme by nature by known method, and obtain first enzyme from this biological cell.This biology is so long as the biology of the enzyme of formation energy catalysis first reaction then without limits, for example can be enumerated the microorganism that comprises real bacterium, eucaryote or archeobacteria.Under the situation of microorganism as the screening object, utilize known method, get final product from the nature separate microorganism, can be from the soil that comprises extreme environments such as high temperature, low temperature, high pressure, acid, alkaline environment, water; Contain the aerial of landing bacterium, ice-nucleus etc.; And obtain in the biological body etc.Particularly the microorganism that separates from hot environment can expect to obtain the first high enzyme of stability from such microorganism.In addition, microorganism can also be bought from strain libraries such as ATCC, DSM.The microorganism that separates carries out pure separation through known method such as Method of Limited Dilution method, the single bacterium colonies of formation, carry out pure culture with basal medium, LB nutrient culture media, broth bouillon etc., confirm to have or not in this culture the enzymatic activity that can carry out phosphorylation to mizoribine and/or Ribavirin, can screen the biology that forms first enzyme thus.In addition, also can be obtained microorganism, be induced generation first enzyme etc. to nutrient culture media interpolation mizoribine and/or Ribavirin by the enrichment culture of using mizoribine and/or Ribavirin, these be the methods that can improve screening effeciency.According to non-patent literature 1 disclosed method etc., those skilled in the art can easily confirm to have or not first enzyme.
The evaluation of strain separated can (Suzuki Ken one bright grade, microorganism divide Class Tong to Ding the biological gimmick The of experiment method-Fen Left Den credit center To (the classification identification experiment method of microorganism-based on the molecular biological method of molecular genetics) according to the experiment book, Springer-Verlag Tokyo) etc. carry out, in addition, can also use commercially available various identification of strains kits (Japanese BIOMERIEUX society etc.), also can entrust evaluations such as juridical person's japanese food analytic centre (the wooden from generation to generation raised path between farm fields of Tokyo Shibuya district unit 52 kinds No. 1).
The natural microbial of formation first enzyme that so obtains can also further be handled with medicaments such as NTG, ultraviolet ray and/or radioactive ray and make variant.The throughput rate that can improve first enzyme that this variant has, the variant that can form first enzyme that has also may form the variant of character such as having excellent stability, throughput rate, reactivity.
In addition, can use the first above-mentioned enzyme or form the natural microbial of this first enzyme, obtain base sequence or its information of resulting this first enzyme of coding by protein sequence, dna sequence dna or known gene engineering method.
First enzyme of the present invention also can obtain by known bioinformatics method.Sequence table sequence numbering 1 and sequence table sequence numbering 2 are amino acid sequence and base sequences of first enzyme of the middle preferred examples of operation (1) as assay method of the present invention, so can carry out homology retrieval with blast search etc. to these sequences at databases such as NCBI, EMBL, GenomeNet in the mode of inquiry (query), thereby obtain to have and sequence table sequence numbering 1 or sequence table sequence numbering 2 are consistent or the similarly information or the gene information of the biology of amino acid sequences or the protein of base sequence or the possibility that gene promptly has generation first enzyme.
The manufacture method of this enzyme comprises the operation of this enzyme that acquisition forms based on the information of the base sequence of the natural microbial of formation first enzyme that utilizes above-mentioned method to obtain, first enzyme or base sequence, utilizes the manufacture method of this enzyme can obtain this first enzyme.As the operation that forms this first enzyme, can enumerate the synthetic system of acellular enzyme of the base sequence that for example contains this enzyme of encoding, preferably enumerate the cell that uses the base sequence that contains this enzyme of encode operation, use the natural microbial that forms this enzyme etc. have this enzyme of coding base sequence microorganism operation, use the operation of the transformant that imports the base sequence that this enzyme of encoding is arranged etc.
Employing contains under the situation of the synthetic system of acellular enzyme of base sequence of first enzyme of encoding, and the base sequence of above-mentioned first enzyme of coding is used to derive from wheat embryo, derives from Escherichia coli, derives from the netted red blood cell of rabbit or derives from the synthetic system of known acellular enzyme such as insect cell get final product.
In addition, adopt to use under the situation of operation of cell of the base sequence that contains this enzyme of encode, for example, the base sequence of above-mentioned coding first enzyme can be inserted carrier, the importing host microorganism is made transformant, uses this transformant to form this enzyme.
Importing has the transformant of the base sequence of coding first enzyme to comprise that the carrier that will insert this base sequence is that recombinant phage or recombinant plasmid import cell or the microorganism that the host forms.Encoding the base sequence of first enzyme can composite part or all use, and preferably uses known method to obtain the base sequence of coding first enzyme from genetic donor.As genetic donor, then unqualified so long as can form the cell of first enzyme, preferably the natural microorganism that filters out with above-mentioned screening technique, comprise biology with formation first enzyme of the biology of bioinformatics method acquired information.
Carrier as the base sequence that inserts coding first enzyme, bacteriophage or the plasmid that preferably in the host microorganism body, can restrain oneself being used for genetic recombination of propagation and make up, as phage vector, for example, can use λ gt λ C, λ gt λ B etc. to belong under the situation that colibacillary microorganism is the host.In addition, as plasmid vector, for example be under host's the situation with Escherichia coli, can use the pET carrier of Novagen society or pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pINI, BluescriptKS+; With the hay bacillus is under host's the situation, can use pWH1520, pUB110, pKH300PLK; With actinomyces is under host's the situation, can use pIJ680, pIJ702; With yeast particularly S. cervisiae (Saccharomyces cerevisiae) be under host's the situation, can use YRp7, pYC1, YEp13 etc.Among the present invention, preferably be host's plasmid vector with Escherichia coli.Promoter then is not particularly limited so long as can express in the host.
Utilize restriction enzyme to cut off above-mentioned carrier, the end that makes its generation is identical with the base sequence end that restriction enzyme generated of base sequence by being used to cut off coding first enzyme, make carrier segments, according to conventional methods, the fragment of base sequence of coding first enzyme is connected with dna ligase with carrier segments, the base sequence of coding first enzyme is inserted the purpose carrier, make recombinant phage or recombinant plasmid.
As the host who imports recombinant plasmid, proliferating cells or microorganism get final product so long as recombinant plasmid can be stablized and restrain oneself, for example host microorganism is when belonging to colibacillary microorganism, can use the Escherichia coli B strain, K strain, C strain or their lysogenic bacteria that comprise e. coli bl21, Escherichia coli DH1, e. coli jm109, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli C600 etc.In addition, host microorganism is when belonging to the microorganism of bacillus, can use hay bacillus, bacillus megaterium (Bacillus megaterium) etc.; Host microorganism is when belonging to actinomycetic microorganism, can use muta lead mycillin (Streptomyces lividans) TK24 etc.; Host microorganism is when belonging to the microorganism of S. cervisiae, can use S. cervisiae INVSC1 etc.The present invention preferably is host microorganism with Escherichia coli.
As using the transformant that imports the base sequence that coding first enzyme is arranged to form preferred other examples of this enzyme, can enumerate the method (No. 3944577 communique of Japan's special permission, No. 3793812 communique of Japan's special permission) of the recombinase that forms the Rhod bacterium.Specifically, provided following method: the base sequence that in the plasmid vectors such as pTip QC1, pTip QC2 that are fit to low temperature formation recombinase, inserts coding first enzyme, this recombinant plasmid is imported lysozyme susceptibility microorganism, form transformant, thereby use this transformant.As lysozyme susceptibility microorganism, preferably belong to the microorganism (No. 3876310 communique of Japan's special permission) of Rhod.
Form the natural microbial of first enzyme, the condition of culture that imports the transformant etc. of the base sequence that this enzyme of coding is arranged and can consider that its nutrition physiology character selects, carry out Liquid Culture usually, the industrial deep ventilation stir culture of carrying out is favourable.As the nutrient source of nutrient culture media, can be extensive use of the material that is generally used for microbe culture.Cultivation temperature can suitably change as host's development of microorganisms, the scope that can form first enzyme, and when the host was Escherichia coli, lowest temperature was more than 10 ℃, was preferably more than 20 ℃, more preferably more than 25 ℃; On be limited to below 45 ℃, be preferably below 42 ℃, more preferably below 38 ℃.When the host is actinomyces, be limited to more than 4 ℃ down, be preferably more than 10 ℃, more preferably more than 20 ℃; On be limited to below 50 ℃, be preferably below 42 ℃, more preferably below 37 ℃.Condition of culture is more or less different because of condition, estimates that first enzyme reaches the period of the highest formation amount, finishes to cultivate in suitable period to get final product, when the host is Escherichia coli, usually be limited to more than 10 hours under, be preferably more than 12 hours, more preferably more than 17 hours; On be limited to below 60 hours, be preferably below 48 hours, more preferably below 30 hours.When the host is actinomyces, be limited to more than 17 hours under usually, be preferably more than 20 hours, more preferably more than 24 hours; On be limited to below 80 hours, be preferably below 72 hours, more preferably below 48 hours.The pH of nutrient culture media can grow and the scope that forms first enzyme suitably changes on bacterium, and the host is that preferred lower limit is more than the pH5.8 under Escherichia coli, the actinomycetic situation, more preferably more than the pH6.2, on be limited to below the pH8.5, be preferably below the pH7.5.
First enzyme can be by comprising first enzyme that acquisition as above forms the method for operation make, it can directly be the cell etc. that contains simple sterilization or do not have the thalline of sterilization, has simply removed the enzyme that contains impurity of cultivating impurity, clasmatosis thing etc. but preferably make.In addition, the thick enzyme of first enzyme is preferably made enzyme free from foreign meter in fact according to purpose, purposes etc., can enumerate usually and make for example various purity more than 50%, more than 70%, more than 95%.Purity can be passed through known methods such as SDS-PAGE, HPLC and confirm.
Utilize to cultivate the above-mentioned natural microbial that screens, import the microorganism etc. of transformant of the base sequence that this enzyme of coding is arranged and then the method that obtains first enzyme is made under the situation of first enzyme, at first this microorganism etc. is cultivated with nutrient medium, make in the thalline or this enzyme of formation in the nutrient solution, in thalline, form under the situation of this enzyme, after cultivating end, from resulting culture, collect thalline by means such as filtration or centrifugings.Next this thalline is destroyed with enzyme methods such as mechanical means or lysozymes, and add EDTA and/or suitable surfactant etc. as required, concentrate or do not concentrate under the condition of this enzyme, implement to utilize the precipitation classification of organic solvents such as acetone, methyl alcohol, ethanol; Utilize the salting out method of ammonium sulfate, salt etc. etc., thereby first enzyme precipitation is reclaimed.As required, this sediment is dialysed, behind the isoelectric precipitation, can be handled by adsorption chromatographies such as gel filtration, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, obtain first enzyme.In addition, but these methods of appropriate combination also.In addition, form in nutrient solution under the situation of enzyme of the present invention, remove thalline by means such as filtration or centrifugings from culture, processing identical when nutrient solution is carried out being formed in the thalline with the enzyme of the invention described above gets final product.
For first enzyme that so obtains, add or do not add various salts, carbohydrate, enzyme, lipid, surfactant etc. as stabilizing agent or excipient, can obtain liquid or the first solid-state enzyme by methods such as ultrafiltration and concentration, freeze dryings, in addition, suitable carrying out under the cryodesiccated situation can be added sucrose about 0.5~10%, sweet mellow wine, salt, albumin etc. as stabilizing agent or excipient.
The typical case that forms the natural microbial of first enzyme of the present invention is Thailand bulkholderia cepasea DSM13276 strain.The typical case of the amino acid sequence of first enzyme in the operation of assay method of the present invention (1) is the amino acid sequence of sequence table sequence numbering 1.The encode base sequence that example is a sequence table sequence numbering 2 of base sequence of this amino acid sequence.According to H.S.Stanley etc., BMC Genomics, 6 volumes, 174 pages, 2005 deliver content, this base sequence has been found out in full genome parsing by Thailand bulkholderia cepasea DSM13276 strain, but never illustrating the character of the enzyme of this base sequence coding so far, only is to infer that simply it is similar to ribokinase.That is, the amino acid sequence of sequence table sequence numbering 1 of in the past not knowing the alkali yl coding of sequence table sequence numbering 2 fully has catalytic action to the reaction at mizoribine and/or Ribavirin.
Manufacture method to second enzyme of the present invention describes below.
Second enzyme of the present invention is identical with the situation of above-mentioned first enzyme, can pass through known method, seeks the natural biological that forms this second enzyme from nature, obtains second enzyme of the present invention from this biological cell.Mizoribine and/or Ribavirin to the inhibition of this second enzyme can by for example relatively the difference of the reaction velocity of this second enzyme under the situation that has and do not exist mizoribine and/or Ribavirin confirmed.Upright guest of phosphoimidazole and/or phosphoric acid Ribavirin to the inhibition of second enzyme can be by second enzyme relatively reaction velocity, for example the difference of the reaction velocity of this second enzyme under the situation that has and do not exist upright guest of phosphoimidazole and/or phosphoric acid Ribavirin is confirmed.Calculate to suppress under the situation that constant compares, the method that can be put down in writing according to the infrastest method of protein enzyme (with reference to revision the 2nd edition, hole tail force one,, Nan Guangtang in 1994) etc. is calculated, is confirmed.Upright guest of phosphoimidazole or phosphoric acid Ribavirin can be made the upright guest of phosphoimidazole of upright guest of phosphoimidazole and/or phosphoric acid Ribavirin and/or the manufacture method manufacturing of phosphoric acid Ribavirin with mizoribine and/or Ribavirin by described later, also can be synthetic by Shuto S. etc., J.Chem.Soc., Perkin Trans.1 volume, 3603 pages, the method put down in writing in 2000.
Preparation method, formation method and the utilization of the base sequence preparation method of second enzyme, the base sequence that bioinformatics method obtains and the information of the biology that produces this enzyme comprises that manufacture method of the method that obtains operation etc. is identical with the situation of above-mentioned first enzyme.
The typical case of second enzyme of the present invention is inosine a 5 ' monophosphate dehydrogenase.The typical case that forms the natural microbial of second enzyme of the present invention is hay bacillus ATCC23857 strain or Oceanobacillusiheyensis DSM14731 strain.The typical case of the amino acid sequence of second enzyme in the operation of assay method of the present invention (2) is the amino acid sequence of sequence table sequence numbering 7 or sequence table sequence numbering 9.The encode base sequence that example is sequence table sequence numbering 8 or sequence table sequence numbering 10 of base sequence of this amino acid sequence.According to Wu T.W. etc., Can.J.Biochem., 51 volumes, 1391 pages, 1973 deliver content, show the base sequence coding inosine 5 ' monophosphate dehydrogenase of sequence table sequence numbering 8, but do not know this inosine 5 ' monophosphate dehydrogenase not to be subjected to mizoribine and the two inhibition of Ribavirin in the past fully and be subjected to the upright guest of phosphoimidazole and phosphoric acid Ribavirin the two suppresses.In addition, according to Takamami H. etc., Nucleic.Acids.Res., 30 volumes, 3927 pages, 2002 deliver content, full genome by Oceanobacillus iheyensis DSM14731 strain is resolved the base sequence of having found out sequence table sequence numbering 10, but never illustrate the character of the enzyme of this base sequence coding so far, do not know still in the past that the amino acid sequence of sequence table sequence numbering 9 of the alkali yl coding of sequence table sequence numbering 10 was inosine 5 ' monophosphate dehydrogenases.In addition, do not know fully in the past that this enzyme was not subjected to mizoribine and the two inhibition of Ribavirin and is subjected to the upright guest of phosphoimidazole and/or phosphoric acid Ribavirin the two suppresses.
Embodiment
Wait based on embodiment below that the present invention will be described, but scope of the present invention should not be construed as and only limits to following embodiment etc.In addition, the technology that is designated as " passing through conventional method " is can be according to the method (Maniatis of for example Maniatis etc., T. etc., molecular cloning, cold spring harbor laboratory, nineteen eighty-two, 1989), subsidiary method of operating is implemented in the infrastest method of above-mentioned protein enzyme, commercially available various enzymes or the kit class.In addition, because the precision of condition determination, use machine etc., measured value given below etc. might change.
In addition, do not particularly point out, the reagent class that the present invention uses can be used the reagent that is easy to from commercially available acquisition as the goods of Wako Pure Chemical Industries, Ltd., SIGMA-ALDRICH company, Takara-Bio Co., Ltd. etc.The DSM bacterial strain can be bought at Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH.The ATCC bacterial strain can be bought at American Type Culture Collection.The manufacturer of reagent, purity etc. are not particularly limited.In addition, the biomaterials such as serum that use among the present invention are commercially available product or the material that has obtained informed consent (having obtained agreement on the basis of the information that application target correctly has been described).
The live vol of first enzyme of the present invention is to measure by following activation measurement 1 or activation measurement 3, and the live vol of second enzyme of the present invention is to measure by following activation measurement 2.The reaction process of the activation measurement 3 of first enzyme of the present invention is few, can measure the accurate activity value simply.The activation measurement 1 of first enzyme of the present invention can be measured as substrate with arbitrary substance.In addition, in this instructions, when needing the live vol of clear and definite first enzyme of the present invention, when not specifying determination method, the live vol that all is converted in the activation measurement 1 is represented.During conversion, can measure an identical corpse or other object for laboratory examination and chemical testing, convert according to the ratio of the variable quantity of this value with each assay method.Because the precision of condition determination, use machine etc., the ratio of variable quantity might change, and for example determination method 1: determination method 3=0.7:3.0.
[activation measurement 1]
[reaction reagent mixed liquor]
20mM Tris/HCl pH of buffer 7.5
20mM glucose
1mM ATP(pH7)
The 1mM magnesium chloride
1mM NADP
50mM KCl
5U/ml glucose-6-phosphate dehydrogenase (G6PD) (Asahi Kasei Pharma Corp's manufacturing)
5U/ml ADP dependence HK (Asahi Kasei Pharma Corp's manufacturing)
The 1mM mizoribine
In the 1.0cm of quartz system cuvette, measure 1.3ml reaction reagent mixed liquor, preheat 2 minutes at 37 ℃.Add 0.03ml and be diluted to first enzyme solutions of suitable concentration, mix, begin reaction at 37 ℃ through 20mM Tris/HCl pH of buffer 7.5.After the reaction beginning, measure the absorbance at 340nm place, obtain light absorption change per 1 minute in the linear response.The light absorption change note of obtaining is made As/min, will replace the blind check note of first enzyme solutions to make Ab/min, utilize (formula 2) to calculate enzymatic activity (U/ml) with Purified Water.
(formula 2)
Enzymatic activity (U/ml)=(As/min-Ab/min)/6.22} * 1.33/0.03 * extension rate
[activation measurement 2]
[reaction reagent mixed liquor]
50mM Tris/HCl pH of buffer 8.0
2mM inosine 5 ' one phosphoric acid
2mM NAD、50mM KCl
In the 1.0cm of quartz system cuvette, measure 2ml reaction reagent mixed liquor, preheat 2 minutes at 37 ℃.Add 0.03ml and be diluted to second enzyme solutions of suitable concentration, mix, begin reaction at 37 ℃ through 50mM Tris/HCl pH of buffer 8.0.After the reaction beginning, measure the absorbance at 340nm place, obtain light absorption change per 1 minute in the linear response.The light absorption change note of obtaining is made As/min, will replace the blind check note of second enzyme solutions to make Ab/min, utilize (formula 3) to calculate enzymatic activity (U/ml) with Purified Water.
(formula 3)
Enzymatic activity (U/ml)=(As/min-Ab/min)/6.22} * 2.03/0.03 * extension rate
[activation measurement 3]
[reaction reagent mixed liquor 1]
30mM kaliumphosphate buffer pH7.0
10mM ATP(pH7)
The 20mM magnesium chloride
The 5mM inosine
50mM potassium chloride
[reaction reagent mixed liquor 2]
100mM Tris/HCl pH of buffer 9.0
The BsuIMPDH of 13U/ml embodiment 33 preparations
20mM NAD
50mM potassium chloride
1mM DTT
200mM EDTA(pH9)
In the 1.0cm of quartz system cuvette, measure 1.0ml reaction reagent mixed liquor, preheat 2 minutes at 37 ℃.Add 0.01ml through be diluted to the enzyme solutions of the present invention of suitable concentration with 30mM kaliumphosphate buffer pH7.0, mix, begin reaction at 37 ℃.The reaction beginning mixed the hydrochloric acid of 2ml 0.1N after 5 minutes, finished reaction, measured the absorbance at 550nm place, obtained light absorption change.The light absorption change note of obtaining is made As/min, will replace the blind check note of first enzyme solutions to make Ab/min, utilize (formula 4) to calculate enzymatic activity (U/ml) with Purified Water.
(formula 4)
Enzymatic activity (U/ml)=(As/min-Ab/min)/19} * 3.01/0.01 * extension rate
[determination of protein concentration]
The protein concentration of first enzyme of the present invention and second enzyme etc. uses the protein quantification kit of Bio-Rad society to measure by conventional method, is that standard is calculated with BSA.
[embodiment 1]
<can carry out the searching 1 of the enzyme of phosphorylation to mizoribine 〉
The microorganism that will separate from muck by conventional method was cultivated 17 hours at 37 ℃ with the 100mlLB nutrient culture media that contain 0.5% inosine, obtained thalline through centrifuging.The 10mM Tris/HCl pH of buffer 7.5 of thalline with 10ml suspended, carry out ultrasonic disruption, obtain crude enzyme liquid through centrifuging.In the following reactant liquor of 500 μ l, add 10 μ l crude enzyme liquids, 37 ℃ of reactions 30 minutes., 25 μ l are separated 10 times of reacted reactant liquor dilutions with Purified Water with HPLC.HPLC uses the Asahipak GS-320 of clear and electrician society system, mobile phase is 200mM sodium phosphate buffer pH3.0, flow velocity is 0.5ml/min, implement to separate in room temperature, monitor the extinction of 260nm, detecting the activity that can carry out phosphorylation to mizoribine is the activity of first enzyme of the present invention, so screen this microorganism for forming the microorganism of first enzyme.Make the above-mentioned crude enzyme liquid of 10ml be adsorbed in Q sep.FF through 8.0 equilibratings of the Tris/HCl of 10mM pH of buffer (General Electric medical treatment bio-science society makes).After fully cleaning with the Tris/HCl pH of buffer 8.0 of 10mM, use contain 0 and the Tris/HCl pH of buffer 8.0 of the 10mM of the KCl of 0.5M carry out linear gradient elution.In active component, add ammonium sulfate, making its ultimate density is 15%, make its Phenyl sep.FF that is adsorbed in 10mM kaliumphosphate buffer pH7.5 equilibrating (General Electric medical treatment bio-science society make), use the 10mM kaliumphosphate buffer pH7.5 that contains 15% and 0% ammonium sulfate to carry out linear gradient elution through containing 15% ammonium sulfate.Active component is used after the G-25 of 10mM kaliumphosphate buffer pH7.5 equilibrating (General Electric medical treatment bio-science society make) desalination, makes it be adsorbed in ResourceQ (General Electric medical treatment bio-science society makes) through 10mM kaliumphosphate buffer pH7.5 equilibrating.After fully cleaning with 10mM kaliumphosphate buffer pH7.5, use contain 0 and the 10mM kaliumphosphate buffer pH7.5 of the KCl of 0.5M carry out linear gradient elution.The G-25 desalination of active component through 10mM kaliumphosphate buffer pH7.0 equilibrating obtains first enzyme.This enzyme is separated with SDS-PAGE, major protein is inhaled be imprinted on the pvdf membrane, carry out protein sequencing, the result has proved that first enzyme of the present invention that present embodiment obtains is colibacillary guanosine inosine kinase.The specific activity of first enzyme that present embodiment obtains is below the 0.01U/mg.So, method according to H.Kawasaki etc., Biosci.Biotechnol.Biochem., 64 volumes, record in 972 pages, 2000, the transformant of the great expression guanosine inosine kinase of preparation e. coli jm109, obtain this guanosine inosine kinase, detection can be carried out the enzymatic activity of phosphorylation to mizoribine, and the result has obtained result same as described above.
[reactant liquor] 10mM Tris/HCl pH of buffer 7.5,30mM KCl, 5mM magnesium chloride, 5mM ATP pH7,1mM DTT, 1mM mizoribine.
[embodiment 2]
<can carry out the searching 2 of the enzyme of phosphorylation to mizoribine 〉
According to Thomas Hansen etc., Extremophiles, 11 volumes, 105-114 page or leaf, the method put down in writing in 2007, preparation derives from phosphofructokinase-B (Phosphofructokinase-B) of Methanococcus jannaschii.Specifically, as template, use the primer of sequence table sequence numbering 13 and sequence table sequence numbering 14 to carry out pcr amplification, the pcr amplification gene is connected in pET17b, transform in e. coli bl21 (DE3) with Methanococcus jannaschii DSM2661 strain.With transformant LB medium culture,, use method refining this enzyme identical with the foregoing description 1 with the phosphofructokinase-B of this gene code of IPTG abduction delivering.By the HPLC identical with the foregoing description 1, confirming refining enzyme, to have the enzymatic activity that can carry out phosphorylation to mizoribine be the activity of first enzyme of the present invention.The specific activity of first enzyme that present embodiment obtains is about 0.02U/mg.
[embodiment 3]
<can carry out the searching 3 of the enzyme of phosphorylation to mizoribine 〉
As template, use the primer of sequence table sequence numbering 15 and sequence table sequence numbering 16 to carry out pcr amplification with the ancient green-ball bacterium DSM4304 strain of glimmering, the pcr amplification gene is connected in pET21a (+), transform in e. coli bl21 (DE3).With transformant LB medium culture,, use method refining this enzyme identical with the foregoing description 1 with the enzyme of this gene code of IPTG abduction delivering.By the HPLC identical with embodiment 1, confirming refining enzyme, to have the enzymatic activity that can carry out phosphorylation to mizoribine be the activity of first enzyme of the present invention.The specific activity of first enzyme that present embodiment obtains is below the 0.01U/mg.
[embodiment 4]
<can carry out the searching 4 of the enzyme of phosphorylation to mizoribine 〉
With reference to the method for B.Sahin etc., Eur.J.Biochem, 271 volumes, record in 3547 pages, 2004, preparation derives from the adenosine kinase of mouse.Specifically, as template, use the primer of sequence table sequence numbering 17 and sequence table sequence numbering 18 to carry out pcr amplification, the pcr amplification gene is connected in pET21a (+), transform in e. coli bl21 (DE3) with the cDNA (Clontech society) of mouse brain.With transformant LB medium culture,, use method refining this enzyme identical with the foregoing description 1 with the enzyme of this gene code of IPTG abduction delivering.By the HPLC identical with embodiment 1, confirming refining enzyme, to have the enzymatic activity that can carry out phosphorylation to mizoribine be the activity of first enzyme of the present invention.The specific activity of first enzyme that present embodiment obtains is below the 0.01U/mg, and the reaction velocity of first enzyme that obtains with embodiment 2 is compared, reaction velocity be about its 1/2nd.In addition, derive from the adenosine kinase of mouse and the homogeny approximate about 90% of the amino acid sequence of the adenosine kinase that derives from the people, preserved the amino acid in activated centre, so infer the character derive from adenosine kinase of mouse and the adenosine kinase that derives from the people identical (Structure,, 6 volumes, 183-193 page or leaf in 1998 etc.).That is, the adenosine kinase that derives from the people also can similarly become first enzyme of the present invention with the adenosine kinase that derives from mouse, and specific activity is below the 0.01U/mg by inference, but the reaction velocity of first enzyme that embodiment 2 obtains is good, is about two times.
[embodiment 5]
<can carry out the searching 5 of the enzyme of phosphorylation to mizoribine 〉
Thailand bulkholderia cepasea DSM13276 strain was cultivated 2 days at 30 ℃ with the 100mlLB nutrient culture media that contains 0.5% inosine, carry out centrifuging, obtain thalline.The 10mM Tris/HCl pH of buffer 7.5 of thalline with 10ml suspended, carry out ultrasonic disruption, obtain crude enzyme liquid through centrifuging.By the HPLC identical with embodiment 1, confirming crude enzyme liquid, to have the enzymatic activity that can carry out phosphorylation to mizoribine be the activity of first enzyme of the present invention.
[embodiment 6]
The extraction of<DNA 〉
Use the LB nutrient culture media, cultivate Thailand bulkholderia cepasea DSM13276 strain 2 days at 30 ℃, to the thalline through collecting bacterium with the 1mg/ml lysozyme soln that contains 50mM Tris/HCl pH of buffer 8.0,50mM EDTA, 15% sucrose in 37 ℃ handle 10 minutes after, add SDS, making its ultimate density is 0.25%, the dissolving thalline.In addition, add the 1:1 mixed liquor of the phenol/methenyl choloride of equivalent, stirs after 30 minutes, carry out centrifuging (12,000rpm, 15 minutes), the recovery water layer.After in the water layer that reclaims, mixing the 3M sodium acetate (pH5.5) of 1/10th amounts, add the ethanol of 2 times of amounts lightly, roll genomic DNA with glass bar and separate.Separated genomic DNA is dissolved in 10mM Tris/HCl pH of buffer 8.0, the 1mM EDTA aqueous solution (following note is sometimes made TE), adds an amount of RNase A,, decompose and mix the RNA that exists 37 ℃ of insulations 1 hour.Next, add the 1:1 mixed liquor of the phenol/methenyl choloride of equivalent, carry out processing same as described above, divide the water intaking layer.In the water layer that branch is got, add the 3M sodium acetates (pH5.5) of 1/10th amounts and the ethanol of 2 times of amounts, with said method isolation of genomic DNA once more.This chromosome is dissolved among the TE, adds the saturated phenol of TE and the 1:1 mixed liquor of methenyl choloride, after will all suspending, carry out identical centrifuging repeatedly, the upper strata is transferred to once more other container.In the upper strata of this separation, add 3M sodium acetate buffer (pH5.5) and ethanol, stir the back, carry out centrifuging (2,000G, 4 ℃, 15 minutes) then, the chromosome of precipitation is cleaned with 75% ethanol, carry out drying under reduced pressure-70 ℃ of coolings 5 minutes.By above operation, the DNA standard items of Thailand bulkholderia cepasea DSM13276 strain have been obtained.
[embodiment 7]
<utilize the PCR method to carry out the amplification of gene shown in the sequence table sequence numbering 2 〉
The primer of implementation sequence table sequence numbering 3 and sequence table sequence numbering 4 is so that at pTip QC1 or the multiple clone site NdeI of pTip QC2 (No. 3944577 communique of Japan's special permission and No. 3793812 communique of Japan's special permission) and the gene of BamHI position insetion sequence table sequence numbering 2.PCR uses KOD archaeal dna polymerase (Japan spins society and makes).The PCR condition is according to the method for operation instructions record.The PCR product of resulting about 1.0kbp is refining according to a conventional method.
[embodiment 8]
<with being connected of expression vector
According to conventional method, with NdeI and BamHI PCR product refining among the embodiment 7 is carried out restriction enzyme and handle, make the insertion fragment.To be connected with conventional method with the pTip QC1 or the pTip QC2 that carry out with NdeI and BamHI making with extra care after restriction enzyme is handled through the refining insertion fragment of conventional method, make pTip QC1/BthNK and pTip QC2/BthNK.By conventional method pTip QC1/BthNK and pTip QC2/BthNK are imported bacillus coli DH 5 alpha, transform, the insertion fragment sequence that confirms by the refining recombinant plasmid of the positive colony that utilizes the bacterium colony direct PCR method to obtain by dna sequencing is correct.
[embodiment 9]
<pTip QC1/BthNK and the conversion of pTip QC2/BthNK in Rhodococcus erythropolis 〉
PTip QC1/BthNK and the conversion of pTip QC2/BthNK in Rhodococcus erythropolis are to utilize electroporation (electroporation) to implement according to the method that TOHKEMY 2004-073116 communique etc. is put down in writing.Electroporation conditions is as follows.Melt competent cell, mix, put into the cup special of 2mm, under the condition of 7.5mS, 2500V, 25 μ F, 400 Ω, apply pulse with pTipQC1/BthNK or pTip QC2/BthNK.
[embodiment 10]
<express and confirm
The transformant that embodiment 9 is obtained is inoculated in the LB nutrient culture media that contains 34 μ g/ml chloromycetin, cultivates 2 days at 30 ℃, makes seed.At the above-mentioned seed of LB inoculation of medium 1/10 amount that contains 34 μ g/ml chloromycetin and 1 μ g/ml thiostrepton, cultivated 1 day at 30 ℃.The centrifuging nutrient solution collects bacterium, is suspended in the 20mM Tris/HCl pH of buffer 7.5 of 1/5 times of amount of nutrient solution, carries out ultrasonic disruption, through centrifuging, with the supernatant that obtains as crude enzyme liquid.The SDS-PAGE of crude enzyme liquid the results are shown in Figure 1, confirms the great expression of enzyme.In addition, the middle arrow of arrow and Fig. 1 (B) is respectively first enzyme (BthNK) (can carry out the enzyme of phosphorylation to mizoribine) of expressing in the transformant of pTip QC1/BthNK, pTip QC2/BthNK among Fig. 1 (A).
[embodiment 11]
<by the refining enzyme that can carry out phosphorylation of pTip QC1/BthNK transformant〉to mizoribine
Make among the embodiment 10 and to cultivate the resulting crude enzyme liquid of pTip QC1/BthNK transformant and be adsorbed in Cherating Sepharose FF (General Electric medical treatment bio-science society makes) with the 50mM phosphate buffer pH8.0 equilibrating that contains 0.1M nickel chloride and 0.3M sodium chloride.After fully cleaning with the phosphate buffer pH8.0 of the 50mM that contains 0.3M sodium chloride, fully clean with the 50mM phosphate buffer pH6.0 that contains 10% glycerine and 0.3M sodium chloride.With the 50mM phosphate buffer pH6.0 wash-out that contains 0.4M imidazoles, 10% glycerine and 0.3M sodium chloride.Active component obtains first enzyme (BthNK) (can carry out the enzyme of phosphorylation to mizoribine) with the 30mM phosphate buffer pH7.0 dialysis that contains 0.03M sodium chloride, 0.05M KCl and 1mM DTT.The specific activity of the BthNK that present embodiment obtains is about 0.04U/mg.The cultivation of 1L obtains about 80mg BthNK.
[embodiment 12]
<by the refining enzyme that can carry out phosphorylation of pTip QC2/BthNK transformant〉to mizoribine
Make the Q sep.BB (General Electric medical treatment bio-science society makes) that cultivates the resulting crude enzyme liquid of pTip QC2/BthNK transformant among the embodiment 10 and be adsorbed in Tris/HCl pH of buffer 8.0 equilibratings of using 10mM.After fully cleaning with the Tris/HCl pH of buffer 8.0 of 10mM, use contain 0 and the Tris/HCl pH of buffer 8.0 of the 10mM of 0.5M KCl carry out linear gradient elution.In active component, add ammonium sulfate, making its ultimate density is 15%, make active component be adsorbed in Phenyl sep.FF, use the 10mM kaliumphosphate buffer pH7.5 that contains 15% and 0% ammonium sulfate to carry out linear gradient elution with the 10mM kaliumphosphate buffer pH7.5 equilibrating that contains 15% ammonium sulfate.Active component is used after the G-25 desalination of 10mM kaliumphosphate buffer pH7.5 equilibrating, makes it be adsorbed in the DEAE sep.FF (General Electric medical treatment bio-science society makes) that uses 10mM kaliumphosphate buffer pH7.5 equilibrating.After fully cleaning with 10mM kaliumphosphate buffer pH7.5, use contain 0 and the 10mM kaliumphosphate buffer pH7.5 of 0.5M KCl carry out linear gradient elution.The G-25 desalination of active component through 10mM kaliumphosphate buffer pH7.0 equilibrating obtains first enzyme (BthNK) (can carry out the enzyme of phosphorylation to mizoribine).The specific activity of the BthNK that present embodiment obtains is about 0.04U/mg.The cultivation of 1L obtains about 50mg BthNK.
[embodiment 13]
<used the pET system can carry out the expression of the enzyme of phosphorylation to mizoribine
By conventional method the insertion fragment that embodiment 7 obtains is connected with the pET21a (+) that carries out with NdeI and BamHI making with extra care after restriction enzyme is handled, makes pET21a (+)/BthNK.By conventional method pET21a (+)/BthNK is imported e. coli bl21 (DE3), transform, the insertion fragment sequence that confirms by the refining recombinant plasmid of the positive colony that utilizes the bacterium colony direct PCR method to obtain by dna sequencing is correct.Resulting transformant is inoculated in the LB nutrient culture media that contains 50 μ g/ml ampicillins.Cultivated 1 day at 30 ℃, make seed.At the above-mentioned seed of LB inoculation of medium 1/100 amount that contains 50 μ g/ml ampicillins, cultivated one day at 30 ℃.Add IPTG, making the concentration in the nutrient solution is 1mM, further cultivates 3 hours at 30 ℃.Nutrient solution is carried out centrifuging and collects bacterium, be suspended in the 20mM Tris/HCl pH of buffer 8.5 of 1/5 times of amount of nutrient solution, carry out ultrasonic disruption, through centrifuging, with the supernatant that obtains as crude enzyme liquid.The SDS-PAGE of crude enzyme liquid the results are shown in Figure 2, confirms the great expression of enzyme.In addition, arrow is first enzyme of the present invention (can carry out the enzyme of phosphorylation to mizoribine) among the figure.Crude enzyme liquid is refining with the method identical with embodiment 12.The specific activity of first enzyme (BthNK) that present embodiment obtains (can carry out the enzyme of phosphorylation to mizoribine) is about 0.04U/mg.The cultivation of 1L obtains about 50mgBthNK.
[embodiment 14]
The upright guest's of the phosphorylation method of<mizoribine and phosphoimidazole manufacturing 〉
In 500 μ l embodiment, 1 described reactant liquor, add the BthNK of 10 μ l, 37 ℃ of reactions 30 minutes with preparation among the embodiment 12 of 10mM Tris/HCl pH of buffer 7.5 dilutions.By the reactant liquor behind the HPLC separating reaction identical with embodiment 1.Reacted chromatogram is seen Fig. 3 (C), and the chromatogram before the reaction is seen Fig. 3 (B).Fig. 3 (A) separates the chromatogram that obtains with HPLC to the upright guest of the phosphoimidazole of standard items under identical HPLC condition.The BthNK that confirms preparation among the embodiment 12 has the enzymatic activity that can carry out phosphorylation to mizoribine.The BthNK that equally, also confirms preparation among the embodiment 13 has the enzymatic activity that can carry out phosphorylation to mizoribine.
[embodiment 15]
<substrate specificity 〉
Use is by the BthNK of embodiment 12 preparations.Use each substrate in the table 1 to replace mizoribine, utilize above-mentioned activation measurement 1 to measure the relative activity of BthNK.Table 1 provides the substrate specificity that has compared relative activity.
[table 1]
Substrate | Relative activity (%) |
Do not have | 0 |
Mizoribine | 11 |
|
1 |
Guanosine | 19 |
|
0 |
Adenosine | 81 |
The 2- |
5 |
|
0 |
Uridine | 2 |
D-ribose | 0 |
L- |
0 |
|
0 |
|
0 |
The 2- |
0 |
|
0 |
Galactose | 0 |
Wood sugar | 0 |
N-acetyl- |
0 |
Fructose-6- |
0 |
Man-6- |
0 |
Ribose-5- |
0 |
|
100 |
The result of present embodiment shows, BthNK be first enzyme of the present invention (can carry out the enzyme of phosphorylation) to mizoribine at least in the presence of phosphodonor catalysis nucleosides is become the reaction of phosphoric acid nucleoside.Act on adenosine, inosine and guanosine especially, and (promptly under the condition of present embodiment) do not act on cytidine and xanthosine in fact, do not act on ribose in fact yet.
In addition, the result of present embodiment shows that BthNK is the reaction that first enzyme of the present invention (can carry out the enzyme of phosphorylation to mizoribine) catalysis becomes mizoribine and/or Ribavirin upright guest of phosphoimidazole and/or phosphoric acid Ribavirin.
[embodiment 16]
<optimal pH 〉
Use is by the BthNK of embodiment 12 preparations.Change the damping fluid in the activation measurement 3, carry out determination of activity, measure optimal pH, wherein the scope of pH4.5~6.0 is used citric acid/NaOH (representing with zero among Fig. 4), the scope of pH6.0~7.5 is used kaliumphosphate buffer (using among Fig. 4 ● expression), the scope of pH7.0~9.0 is used Tris/HCl damping fluid (representing with △ among Fig. 4), and pH9.5 uses glycocoll/NaOH damping fluid (representing with among Fig. 4).It is 100% relative activity (%) that Fig. 4 has provided with maximum activity.BthNK shows the scope of the optimal pH of maximum activity in pH5.5~7.5.
[embodiment 17]
<pH stability 〉
Use is measured pH stability by the BthNK of embodiment 12 preparations.BthNK is diluted in the following damping fluid of 100mM by pH, making concentration is 0.475mg/ml, heated processing after 3 hours at 37 ℃, measure the remaining activity of BthNK with activation measurement 3, wherein, the scope of pH4.5~6.0 is used citric acid/NaOH (representing with zero among Fig. 5), the scope of pH6.0~7.5 is used kaliumphosphate buffer (using among Fig. 5 ● expression), the scope of pH7.0~9.0 is used Tris/HCl damping fluid (representing with △ among Fig. 5), and glycocoll/NaOH damping fluid (representing with among Fig. 5) is used in pH9.5~11.0.As shown in Figure 5, BthNK scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%.
[embodiment 18]
<thermal stability 〉
Use is measured thermal stability by the BthNK of embodiment 12 preparations.BthNK is diluted among the 100mM kaliumphosphate buffer pH7.0, and making concentration is 0.475mg/ml, after 20 minutes, measures the remaining activity of BthNK in each heat treatment with activation measurement 3.As shown in Figure 6, BthNK when carrying out 20 minutes thermal treatment for 50 ℃, keeps the activity more than 80% in the aqueous solution of 100mM kaliumphosphate buffer pH7.0.
[embodiment 19]
The molecular weight of<BthNK 〉
As shown in Figure 7, the molecular weight based on sds polyacrylamide gel electrophoresis method (SDS-PAGE) by the BthNK of embodiment 12 preparation is about 34kDa.The calculated value that is obtained by the amino acid sequence of sequence table sequence numbering 1 is 33994.
[embodiment 20]
The operative temperature scope of<BthNK 〉
BthNK with activation measurement 3 is measured by embodiment 12 preparations wherein, changes temperature of reaction 15~70 ℃ scopes, measures BthNK carries out phosphorylation to inosine reaction velocity (Fig. 8).Its result is Arrhenius figure, is shown in Fig. 9.As shown in Figure 8, the scope of operative temperature is 15~70 ℃ at least.In addition, as shown in Figure 9, the energy of activation between 20~45 ℃ is about 64kJ/mol.In addition, T represents absolute temperature among Fig. 9.In addition, as seen from Figure 8, do not improve, it is believed that this is because the result that the decomposition of the stability of the BthNK in the reactant liquor and ATP causes at relative activity more than 50 ℃.
[embodiment 21]
The usability of<metal 〉
In following [potpourri] that used the BthNK for preparing by embodiment 12, use the metallic compound shown in the table 2, measure and utilize this moment BthNK to generate the speed of AMP, with the form that compares it be the results are shown in table 2.
At the 1ml[potpourri] in add adenosine, making its ultimate density is 1mM, at 37 ℃ adenosine is carried out 30 minutes phosphorylations.With 10 times of Purified Water [potpourri] dilutions after with phosphorylation, 25 μ l are separated with HPLC.HPLC uses the Asahipak GS-320 of clear and electrician society system, and mobile phase is used 200mM sodium phosphate buffer pH3.0, and flow velocity is 0.5ml/min, implements to separate in room temperature, with the growing amount of the absorptiometry AMP of 260nm.
[potpourri]
50mM Tris/HCl pH of buffer 7.5
2.5mM ATP
Metallic compound shown in the 5mM table 2
0.5mU/ml the BthNK of embodiment 12 preparations
[table 2]
Metallic compound | (%) compares |
|
90 |
|
100 |
Nickel chloride | 86 |
Cobalt chloride | 85 |
BthNK utilizes more than among magnesium ion, cobalt ions, nickel ion or the manganese ion any one at least.
[embodiment 22]
<Vmax and Km 〉
With the BthNK that activation measurement 1 is measured by embodiment 12 preparations, wherein, the adjustment concentration of substrate is the variable concentrations more than 5 between 1/10 times~10 times of Km value, utilizes Lineweaver-Burk graphing method reciprocal, calculates each apparent Vmax and Km value.It the results are shown in Table 3.
[table 3]
Substrate | Vmax(μmol/min/mg) | Km(mM) |
Mizoribine | 0.04 | 0.27 |
Adenosine | 0.19 | 0.02 |
Guanosine | 0.17 | 0.54 |
Inosine | 0.46 | 0.10 |
In addition, utilize additive method to measure Vmax and the Km to inosine by the BthNK of embodiment 12 preparations.
[reaction reagent mixed liquor 1]
30mM kaliumphosphate buffer pH7.0
10mM ATP(pH7)
The 20mM magnesium chloride
The 5mM inosine
50mM KCl
[reaction reagent mixed liquor 2]
100mM Tris/HCl pH of buffer 9.0
The BsuIMPDH of 13U/ml embodiment 33 preparations
20mM NAD
50mM KCl
1mM DTT
200mM EDTA(pH9)
In the 1.0cm of quartz system cuvette, measure 1.0ml reaction reagent mixed liquor 1, preheat 2 minutes at 37 ℃.Add 0.05ml and be diluted to the BthNK of suitable concentration, mix, begin to react at 37 ℃ with 30mM kaliumphosphate buffer pH7.0.Mix 1.0ml reaction reagent mixed liquor 2 after 5 minutes, measure the absorbance at 340nm place, obtain light absorption change.Between 1/10 times~10 times of Km value, adjust inosine concentration and NAD concentration and be the different concentration more than 5, calculate Km and Vmax inosine according to Lineweaver-Burk graphing method reciprocal.Its result is Vmax=1.59 μ mol/min/mg, Km=0.18 μ mol/min/mg.
[embodiment 23]
The phosphorylation method of<nucleosides and the manufacturing of phosphoric acid nucleoside 〉
Use prepares following [composition] phosphoric acid agent as nucleosides by the BthNK of embodiment 12 preparations, implements the phosphorylation method of adenosine, makes AMP (AMP) by adenosine.At the 1ml[composition] in add adenosine, making its ultimate density is 1mM, 37 ℃ of phosphorylation methods of implementing 30 minutes adenosines.To implement 10 times of [composition] dilutions after the phosphorylation method of adenosine with Purified Water, 25 μ l will be separated with HPLC.Similarly implement HPLC with the method for record among the embodiment 1.Chromatogram after the phosphorylation method of enforcement adenosine is seen Figure 10 (C), and the chromatogram before implementing is seen Figure 10 (B).Figure 10 (A) is that ATP, ADP, AMP and the adenosine composition to standard items separates the chromatogram that obtains with HPLC under identical HPLC condition.With having used the phosphorylation method that to implement adenosine to the phosphoric acid agent of nucleosides that mizoribine carries out the enzyme of phosphorylation of the present invention, can make AMP (AMP) by adenosine.Blind check uses Purified Water to replace being implemented by the BthNK of embodiment 12 preparations, confirms chromatogram and Figure 10 (B) no change before and after implementing.
[composition]
50mM Tris/HCl pH of buffer 7.5
2.5mM ATP
The 5mM magnesium chloride
0.5mU/ml BthNK by embodiment 12 preparations
[embodiment 24]
The mensuration of<nucleosides potpourri 〉
Use is made following [composition] by the BthNK of embodiment 12 preparations, implements the assay method of the potpourri of adenosine, inosine and guanosine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 5 μ l, R1 are 100 μ l[compositions 1], R2 is 100 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is 1 end-point method, reaction in 10 minutes, 0-31, deducts the blank that Purified Water produces.Sample uses the 1:1:1 potpourri of adenosine, inosine and guanosine, will add up to concentration to adjust to the transverse axis scope of Figure 11, and measurement result is seen Figure 11.Utilization has used the composition of BthNK to implement the mensuration of adenosine, inosine and guanosine potpourri.Need to prove, so-called 1 end-point method, 10 minutes reactions, 0-31 refer to one of assay method of measuring machine (the automatic analytical engine of Hitachi's 7080 types), and those skilled in the art can understand by the operational manual of the automatic analytical engine of reference Hitachi 7080 types.
[composition 1]
20mM kaliumphosphate buffer pH7.0
10mM ATP(pH7)
The 20mM magnesium chloride
0.025U/ml BthNK
50mM KCl
[composition 2]
20mM kaliumphosphate buffer pH7.0
20mM glucose
The 20mM magnesium chloride
50mM KCl
5mM NADP
The 5U/ml glucose-6-phosphate dehydrogenase (G6PD)
5U/ml ADP dependence HK
[embodiment 25]
The mensuration of<nucleosides 〉
Use is made following [composition] by the BthNK of embodiment 12 preparations, implements the assay method of adenosine, inosine and guanosine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 3 μ l, R1 are 130 μ l[compositions], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 5 minutes, 7-9, deducts the blank that Purified Water produces.Sample uses adenosine, inosine and guanosine, and concentration is separately adjusted to the concentration range of the transverse axis of Figure 12~Figure 14, and the measurement result of this moment is seen Figure 12~Figure 14.Utilization has used the composition of BthNK to implement the mensuration of adenosine, inosine and guanosine.Need to prove, so-called rate method A (RATE-A), reaction in 5 minutes, 7-9 (hereinafter being 20-22) refer to one of assay method of measuring machine (the automatic analytical engine of Hitachi's 7080 types), and those skilled in the art can understand by the operational manual of the automatic analytical engine of reference Hitachi 7080 types.
[composition]
50mM kaliumphosphate buffer pH7.0
50mM KCl
1mM ATP(pH7)
0.5mM magnesium chloride
0.5mM PEP
0.1mM NADH
The 50U/ml pyruvate kinase
The 15U/ml lactic dehydrogenase
2mM DTT
2.5mU/ml BthNK
[embodiment 26]
The mensuration of<nucleosides 〉
Use is made following [composition] by the BthNK of embodiment 12 preparations, implements the assay method of adenosine, inosine and guanosine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 3 μ l, R1 are 130 μ l[compositions], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 5 minutes, 7-9, deducts the blank that Purified Water produces.Sample uses adenosine, inosine and guanosine, and concentration is separately adjusted to the concentration range of the transverse axis of Figure 15~Figure 17, and the measurement result of this moment is seen Figure 15~Figure 17.Utilization has used the composition of BthNK to implement the mensuration of adenosine, inosine and guanosine.
[composition]:
20mM kaliumphosphate buffer pH7.0,
20mM glucose,
1mM ATP(pH7)、
The 1mM magnesium chloride,
1mM NADP、
50mM KCl、
The 5U/ml glucose-6-phosphate dehydrogenase (G6PD),
5U/ml ADP dependence HK,
0.6mU/ml BthNK。
[embodiment 27]
To by the BthNK of embodiment 11 preparation, carry out above-mentioned physicochemical property<1 simultaneously by the BthNK of embodiment 12 preparations with by the BthNK of embodiment 13 preparations 〉~<4 mensuration, and compare.Results verification has above-mentioned physicochemical property<1 to these 3 kinds of BthNK 〉~<4 enzyme.
[embodiment 28]
The extraction of<DNA 〉
Use contains the nutrient culture media of 2.3% nutrients, 0.2% potato extract, and hay bacillus ATCC23857 strain was cultivated 1 day at 26 ℃, obtains thalline.5g/L peptone, 1g/L yeast extract, 20g/L NaCl, 0.1g/L ferric citrate, 5.9g/L magnesium chloride, 3.24g/L sodium sulphate are dissolved with process water, be mixed with the nutrient culture media of pH7.6, use this nutrient culture media that Oceanobacillusiheyensis DSM14731 strain was cultivated 1 day at 25 ℃, obtain thalline.By operation similarly to Example 5, obtain the DNA standard items of hay bacillus ATCC23857 strain and Oceanobacillusiheyensis DSM14731 strain by these thalline.
[embodiment 29]
<utilize the PCR method to carry out the amplification of gene shown in the sequence table sequence numbering 8 〉
The DNA of the hay bacillus ATCC23857 strain that obtains with embodiment 28 uses the primer of sequence table sequence numbering 19 and sequence table sequence numbering 20 to carry out gene magnification with PCR as template.Use the KOD archaeal dna polymerase to carry out PCR according to conventional method.The PCR product of resulting about 1.5kbp is made with extra care according to conventional method.
[embodiment 30]
<utilize the PCR method to carry out the amplification of gene shown in the sequence table sequence numbering 10 〉
The DNA of the Oceanobacillus iheyensis DSM14731 strain that obtains with embodiment 28 uses the primer of sequence table sequence numbering 21 and sequence table sequence numbering 22 to carry out gene magnification with PCR as template.Use the KOD archaeal dna polymerase to carry out PCR according to conventional method.The PCR product of resulting about 1.5kbp is made with extra care according to conventional method.
[embodiment 31]
<with being connected of expression vector
According to conventional method, with XbaI and SacI PCR product refining among the embodiment 29 is carried out restriction enzyme and handle, make the insertion fragment.According to conventional method, will be connected with the pHSG399 that carries out with XbaI and SacI making with extra care after restriction enzyme is handled through the refining insertion fragment of conventional method, make pHSG399/BsuIMPDH.By conventional method pHSG399/BsuIMPDH is imported Escherichia coli W3110, transform, the insertion fragment sequence that confirms by the refining recombinant plasmid of the positive colony that utilizes the bacterium colony direct PCR method to obtain by dna sequencing is correct.
[embodiment 32]
<with being connected of expression vector
According to conventional method, with XbaI and SacI PCR product refining among the embodiment 29 is carried out restriction enzyme and handle, make the insertion fragment.According to conventional method, will be connected with the pHSG399 that carries out with XbaI and SacI making with extra care after restriction enzyme is handled through the refining insertion fragment of conventional method, make pHSG399/ObIMPDH.By conventional method pHSG399/ObIMPDH is imported Escherichia coli W3110, transform, the insertion fragment sequence that confirms by the refining recombinant plasmid of the positive colony that utilizes the bacterium colony direct PCR method to obtain by dna sequencing is correct.
[embodiment 33]
The acquisition of<BsuIMPDH 〉
The transformant that embodiment 31 is obtained is inoculated in the LB nutrient culture media that contains 34 μ g/ml chloromycetin and 1mM IPTG, cultivates 1 day at 30 ℃.Nutrient solution is carried out centrifuging and collects bacterium, be suspended in the 20mM Tris/HCl pH of buffer 7.5 that contains 1mM DTT of 1/5 times of amount of nutrient solution, carry out ultrasonic disruption, obtain crude enzyme liquid through centrifuging.Make this crude enzyme liquid be adsorbed in the Q sep.BB of 10mM Tris/HCl pH of buffer 8.0 equilibratings through containing 1mM DTT.After fully cleaning with the 10mM Tris/HCl pH of buffer 8.0 that contains 1mM DTT, use contain 1mMDTT and 0 or the 10mM Tris/HCl pH of buffer 8.0 of 0.5M KCl carry out linear gradient elution.The adding ultimate density is 15% ammonium sulfate in active component, make its Phenyl sep.FF that is adsorbed in the 10mM kaliumphosphate buffer pH7.5 equilibrating of ammonium sulfate, use the 10mM kaliumphosphate buffer pH7.5 of the ammonium sulfate that contains 1mM DTT and 15% or 0% to carry out linear gradient elution through containing 1mMDTT and 15%.The G-25 desalination of active component through 10mM kaliumphosphate buffer pH7.5 equilibrating obtains deriving from the inosine 5 ' monophosphate dehydrogenase (BsuIMPDH) of hay bacillus.The cultivation of 1L obtains the BsuIMPDH of about 10mg.
[embodiment 34]
The acquisition of<ObIMPDH 〉
The transformant of using embodiment 32 to obtain is similarly operated with embodiment 33, obtains deriving from the inosine 5 ' monophosphate dehydrogenase (ObIMPDH) of Oceanobacillus iheyensis.The cultivation of 1L obtains the ObIMPDH of about 10mg.
[embodiment 35]
The optimal pH of<BsuIMPDH and ObIMPDH 〉
Measure the BsuIMPDH of preparation among embodiment 33 and the embodiment 34 and the optimal pH of ObIMPDH.Change the damping fluid in the above-mentioned activation measurement 2, carry out determination of activity, measure optimal pH, wherein, the scope of pH4.5~6.0 is used citric acid/NaOH (representing with zero among Figure 18), the scope of pH6.0~7.5 is used kaliumphosphate buffer (using among Figure 18 ● expression), and the scope of pH7.0~9.0 is used Tris/HCl damping fluid (representing with △ among Figure 18), and glycocoll/NaOH (representing with among Figure 18) is used in pH9.5~10.5.It is 100% relative activity (%) that Figure 18 has provided with maximum activity.Figure 18 (A) is BsuIMPDH, (B) is ObIMPDH.BsuIMPDH and ObIMPDH show the scope of the optimal pH of maximum activity in pH8~9.
[embodiment 36]
The pH stability of<BsuIMPDH and ObIMPDH 〉
Measure embodiment 33 and the BsuIMPDH of embodiment 34 preparations and the pH stability of ObIMPDH.BsuIMPDH and ObIMPDH are diluted in the following damping fluid of 100mM by pH, making concentration is 1mg/ml, heated processing after 3 hours at 37 ℃, measure remaining activity with activation measurement 2, wherein, the scope of pH4.5~6.0 is used citric acid/NaOH (representing with zero among Figure 19), the scope of pH6.0~7.5 is used kaliumphosphate buffer (using among Figure 19 ● expression), the scope of pH7.0~9.0 is used Tris/HCl damping fluid (representing with △ among Figure 19), glycocoll/NaOH (representing with among Figure 19) is used in pH9.5~11.0, and sodium phosphate buffer (representing with ■ among Figure 19) is used in pH10.5~12.5.Among Figure 19, (A) being BsuIMPDH, (B) is ObIMPDH.BsuIMPDH and the ObIMPDH scope in pH6~11 under 37 ℃, 3 hours condition keeps the activity more than 70%.
[embodiment 37]
The thermal stability of<BsuIMPDH and ObIMPDH 〉
Measure embodiment 33 and the BsuIMPDH of embodiment 34 preparations and the thermal stability of ObIMPDH.BsuIMPDH and ObIMPDH are diluted among the 50mM kaliumphosphate buffer pH7.0 that contains 1mM DTT, and making concentration is 1mg/ml, after 30 minutes, measures remaining activity with activation measurement 2 in each heat treatment.Among Figure 20, (A) being BsuIMPDH, (B) is ObIMPDH.As shown in figure 20, BsuIMPDH and ObIMPDH when carrying out 30 minutes thermal treatment for 60 ℃, keep the activity more than 85% in the aqueous solution of the 50mM kaliumphosphate buffer pH7 that contains 1mM DTT.
[embodiment 38]
The molecular weight of<BsuIMPDH and ObIMPDH 〉
As shown in figure 21, the BsuIMPDH of preparation and the molecular weight based on the sds polyacrylamide gel electrophoresis method of ObIMPDH are 52~54kDa among embodiment 33 and the embodiment 34.Among Figure 21, (A) being ObIMPDH, (B) is BsuIMPDH.According to the calculated value that the amino acid sequence by sequence table sequence numbering 7 and sequence table sequence numbering 9 obtains, BsuIMPDH is 52990, and ObIMPDH is 52487.
[embodiment 39]
The influence of the activity of<salt pair BsuIMPDH and ObIMPDH 〉
Change the KCl concentration in the above-mentioned activation measurement 2, measure embodiment 33 and the BsuIMPDH of embodiment 34 preparations and the activity of ObIMPDH, its result is usefulness (zero) expression in Figure 22, changes KCl into ammonium chloride and changes result's usefulness (●) expression in Figure 22 that concentration is measured.Among Figure 22, (A) being ObIMPDH, (B) is BsuIMPDH.In the KCl or ammonium chloride of 50~100mM, BsuIMPDH and ObIMPDH's is active maximum.
[embodiment 40]
The kinetic parameter of<BsuIMPDH and ObIMPDH 〉
Use the BsuIMPDH and the ObIMPDH of embodiment 33 and embodiment 34 preparations.Between 1/10 times~10 times of Km, the concentration of NAD in the above-mentioned activation measurement 2 and inosine 5 ' one phosphoric acid (being expressed as IMP in the table) is adjusted into variable concentrations more than 5, Vmax and Km with Lineweaver-Burk graphing method mensuration BsuIMPDH reciprocal and ObIMPDH the results are shown in Table 4.
In the reaction reagent mixed liquor of above-mentioned activation measurement 2, the concentration of adjusting mycophenolic acid (being expressed as MPA in the table) between 1/10 times~10 times of Ki ' is the variable concentrations more than 5, utilize the Ki ' of 2 map mensuration BsuIMPDH and ObIMPDH of Lineweaver-Burk graphing method, the results are shown in Table 4.
In the reaction reagent mixed liquor of above-mentioned activation measurement 2, the concentration of adjusting the upright guest of phosphoimidazole, xanthosine 5 ' one phosphoric acid (being expressed as MZR-P, XMP in the table), ATP, ADP, AMP between 1/10 times~10 times of Ki is the variable concentrations more than 5, Ki with Dixon graphing method mensuration BsuIMPDH and ObIMPDH the results are shown in Table 4.
BsuIMPDH and ObIMPDH are not subjected to the two the inhibition of mizoribine and Ribavirin as a result.
In the inhibition form, UC represents that uncompetitive suppresses, and CO represents competitive the inhibition, and NC represents that noncompetitive suppresses, and CO+NC represents the mixed type that competitiveness suppresses and noncompetitive suppresses.
By the results verification of present embodiment to BsuIMPDH and ObIMPDH be can catalysis second reaction i.e. second enzyme of enzyme.
[table 4]
BsuIMPDH | ObIMPDH | The inhibition form | |
Vmax (μmol/min/mg) | 2.0 | 0.6 | |
Km | |||
NAD(mM) | 1.4 | 0.8 | |
IMP(mM) | 0.1 | 0.1 | |
Ki’ | |||
MPA(mM) | 14 | 12 | UC |
Ki | |||
MZR-P(nM) | 72 | 157 | CO+NC |
XMP(mM) | 0.15 | 0.60 | CO |
ATP(mM) | 1.2 | 11 | CO+NC |
ADP(mM) | 5.0 | 9.2 | CO+NC |
AMP(mM) | 1.6 | 1.4 | CO+NC |
[embodiment 41]
The mensuration 1 of<mizoribine 〉
Use first enzyme of embodiment 2 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 5 μ l, R1 are 100 μ l[compositions 1], R2 is 50 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 23-25.As sample, mizoribine after adjusting, the scope of 0~10 μ M is measured respectively with PBS, and measurement result is seen Figure 23.Utilization has used the composition of first enzyme of embodiment 2 preparations to implement the mensuration of mizoribine.
[composition 1]
30mM kaliumphosphate buffer pH7.0
75mM KCl
5mM ATP(pH7)
The 10mM magnesium chloride
1mM DTT
2mM inosine 5 ' one phosphoric acid
[composition 2]
50mM Tris/HCl pH of buffer 8.5
75mM KCl
10mM NAD
1mM DTT
0.2U/ml the BsuIMPDH of embodiment 33 preparations
[embodiment 42]
The mensuration of<Ribavirin 〉
Use the BthNK of embodiment 12 preparations and the BsuIMPDH of embodiment 33 preparations, make following [composition], implement the assay method of Ribavirin.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 10 μ l, R1 are 200 μ l[compositions 1], R2 is 100 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.As sample, Ribavirin to be measured after the scope adjustment of 0~10mM respectively with PBS, measurement result is seen Figure 24.Utilization has used the composition of the BthNK of embodiment 12 preparations and the BsuIMPDH that embodiment 33 prepares to implement the mensuration of Ribavirin.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
The BthNK of 4U/ml embodiment 12 preparations
0.2U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
30mM NaCl
0.05% sodium azide
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
0.05% sodium azide
[embodiment 43]
The mensuration 2 of<mizoribine 〉
Use the BthNK of embodiment 11 preparations and the BsuIMPDH of embodiment 33 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 2 μ l, R1 are 230 μ l[compositions 1], R2 is 115 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.As sample, mizoribine to be measured after the scope adjustment of 0~40 μ M respectively with PBS, measurement result is seen Figure 25.Utilization has used the composition of the BthNK of embodiment 11 preparations and the BsuIMPDH that embodiment 33 prepares to implement the mensuration of mizoribine.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.3U/ml the BthNK of embodiment 11 preparations
0.29U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 44]
The mensuration 3 of<mizoribine 〉
Use the BthNK of embodiment 11 preparations and the BsuIMPDH of embodiment 33 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 2 μ l, R1 are 230 μ l[compositions 1], R2 is 115 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.The measurement result with the sample of distilled water diluting mizoribine of 0~2 μ M scope is represented with zero in Figure 26, will in Figure 26, be used with the measurement result of the sample of serum (SeronormHuman (Sero society)) dilution ● expression.Utilization has used the composition of the BsuIMPDH of the BthNK of embodiment 11 preparations and embodiment 33 preparations to implement the mizoribine in the distilled water and the mensuration of the mizoribine in the serum.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.3U/ml the BthNK of embodiment 11 preparations
0.06U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
[composition 2]
100mM Tris/HCl pH of buffer 7.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 45]
The mensuration 4 of<mizoribine 〉
Use BthNK and the BsuIMPDH of embodiment 33 preparations or the ObIMPDH of embodiment 34 preparations of embodiment 12 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 2 μ l, R1 are 230 μ l[compositions 1], R2 is 115 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.As sample, with mizoribine with PBS respectively in the scope adjustment of 0~40 μ M, use the situation of the composition 1 of the BsuIMPDH that has wherein used embodiment 33 preparations in Figure 27, to represent, use the situation of the composition 1 of the ObIMPDH that has wherein used embodiment 34 preparations in Figure 27, to use ● expression with zero.Utilization has used the composition of the ObIMPDH of the BsuIMPDH of the BthNK of embodiment 12 preparations and embodiment 33 preparations or embodiment 34 preparations to implement the mensuration of mizoribine.
[composition 1]:
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.3U/ml the BthNK of embodiment 12 preparations
0.12U/ml the ObIMPDH of the BsuIMPDH of embodiment 33 preparations or embodiment 34 preparations
1mM DTT
30mM NaCl
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 46]
The mensuration 5 of<mizoribine 〉
Use the BthNK of embodiment 12 preparations and the BsuIMPDH of embodiment 33 preparations, make following [composition], the influence of research interferences confrontation measured value.Interfere material to use the interference detection A of international reagent society system (to do Wataru チ
The エ StarNetwork A).The mizoribine of dissolving 2 μ g/ml is measured with following [composition] in the serum of cholerythrin C, the cholerythrin F of the transverse axis concentration that is mixed with Figure 28 (A)~(D), chyle, haemoglobin.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 2.2 μ l, R1 are 200 μ l[compositions 1], R2 is 100 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.Utilization has used the BthNK of embodiment 12 preparations and the composition of the BsuIMPDH that embodiment 33 prepares to implement the mensuration of mizoribine and do not interfered the influence of material.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.4U/ml the BthNK of embodiment 12 preparations
0.16U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
30mM NaCl
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 47]
The mensuration 6 of<mizoribine 〉
Use BthNK or the BthNK of embodiment 13 preparations and the BsuIMPDH of embodiment 33 preparations of embodiment 12 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 2.2 μ l, R1 are 200 μ l[compositions 1], R2 is 100 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.As sample, with mizoribine with PBS respectively in the scope adjustment of 0~5 μ g/ml, use the situation of the composition 1 of the BthNK that has wherein used embodiment 12 preparations in Figure 29, to represent, use the situation of the composition 1 of the BthNK that has wherein used embodiment 13 preparations in Figure 29, to use ● expression with zero.Utilization has used the composition of the BthNK of embodiment 12 preparations or the BthNK that embodiment 13 prepares to implement the mensuration of mizoribine.
[composition 1]:
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.4U/ml the BthNK of the BthNK of embodiment 12 preparations or embodiment 13 preparations
0.16U/ml the BsuIMPDH of embodiment 33 preparations
2mM DTT
30mM NaCl
0.05% TX-100
0.05% sodium azide
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
2mM DTT
20mM inosine 5 ' one phosphoric acid
0.05% sodium azide
[embodiment 48]
The mensuration 7 of<mizoribine 〉
Use the BthNK of embodiment 13 preparations and the BsuIMPDH of embodiment 33 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, and condition determination is rate method A, reaction in 10 minutes, 20-22.Parameter is that sample is that 2 μ l, R1 are 200 μ l[compositions 1], R2 is 100 μ l[compositions 2] situation under, measure the sample (Figure 30 (A)) that is adjusted at the scope of 0~7.5 μ g/ml with PBS.Parameter is that sample is that 5 μ l, R1 are 100 μ l[compositions 1], R2 is 50 μ l[compositions 2] situation under, measure the sample (Figure 30 (B)) that is adjusted at the scope of 0~0.5 μ g/ml with PBS.Utilization used embodiment 13 preparations BthNK composition measuring the mizoribine of 0~7.5 μ g/ml scope and 0~0.5 μ g/ml scope.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.4U/ml the BthNK of embodiment 13 preparations
0.12U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
30mM NaCl
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 49]
The mensuration 8 of<mizoribine 〉
Use the BthNK of embodiment 11 preparations and the BsuIMPDH of embodiment 33 preparations, make following [composition], implement the assay method of mizoribine.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, and condition determination is rate method A, reaction in 10 minutes, 20-22.Parameter is as follows: sample is that 5 μ l, R1 are 100 μ l[compositions 1], R2 is 50 μ l[compositions 2].As sample, use in Figure 31 with the situation of serum dilution preparation mizoribine (0~2 μ M) ● expression, the situation of the hematoclasis liquid dilution preparation mizoribine (0~2 μ M) that forms in order to 5 times of dilutions of distilled water haemocyte is represented with zero at Figure 31.Utilization used the BthNK of embodiment 11 preparations and embodiment 33 preparations BsuIMPDH composition measuring the mizoribine of serum dilution of 0~2 μ M scope and the mizoribine of hematoclasis liquid dilution.
[composition 1]
30mM Tris/HCl pH of buffer 7.5
50mM KCl
10mM ATP(pH7)
5mM inosine 5 ' one phosphoric acid
The 20mM magnesium chloride
0.05% TX-100
0.1U/ml the BthNK of embodiment 11 preparations
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
10mM NAD
0.05% TX-100
1mM DTT
0.06U/ml the BsuIMPDH of embodiment 33 preparations
[embodiment 50]
The mensuration 9 of<mizoribine 〉
In 40 corpse or other object for laboratory examination and chemical testing of human serum, add the mizoribine of 0.5~6.9 μ g/ml scope, make sample.For this sample, will use the BsuIMPDH of the BthNK of embodiment 12 preparations and embodiment 33 preparations and use following [composition] to measure mizoribine obtains in the serum result's (mizoribine assay method of the present invention) and measure the result that mizoribine obtains in the serum by the method (utilizing HPLC to measure mizoribine) of using embodiment 1 described HPLC and do to compare.Shown in figure 32, mizoribine assay method of the present invention (X-axis) and the relational expression of utilizing the mizoribine of HPLC to measure (Y-axis) are about Y=1.1X+0.09.Thereby confirm, mizoribine assay method of the present invention with utilize the mizoribine assay method of HPLC to have equal accuracy.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.19U/ml the BthNK of embodiment 12 preparations
0.16U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
30mM NaCl
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 51]
The upright guest's of<phosphoimidazole mensuration 〉
Use the BsuIMPDH of embodiment 33 preparations, make following [composition], implement the upright guest's of phosphoimidazole assay method.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 2 μ l, R1 are 230 μ l[compositions 1], R2 is 115 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.The measurement result with the upright guest's of distilled water diluting phosphoimidazole sample of 0~35 μ M scope is seen Figure 33.Utilization has used the composition of the BsuIMPDH of embodiment 33 preparations to implement the upright guest's of the phosphoimidazole in the distilled water mensuration.
[composition 1]
30mM kaliumphosphate buffer pH7.0
50mM KCl
5mM NAD
0.05% TX-100
0.16U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
[composition 2]
100mM Tris/HCl pH of buffer 7.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 52]
The mensuration 10 of<mizoribine 〉
With 47 corpse or other object for laboratory examination and chemical testing of serum of collecting from the patient who uses mizoribine as sample.To use the BsuIMPDH of the BthNK of embodiment 12 preparations and embodiment 33 preparations and use following [composition] to measure mizoribine obtains in the serum result's (mizoribine assay method of the present invention) and measure the result that mizoribine obtains in the serum and do to compare by Journal of chromatography, 432 volumes,, the method (utilizing HPLC to measure mizoribine) of the use HPLC of 340-345 page or leaf record in 1988.As shown in figure 34, mizoribine assay method of the present invention (Y-axis) is about Y=1.1X+0.06 with the relational expression of the mizoribine mensuration (X-axis) of utilizing HPLC.Thereby confirm, mizoribine assay method of the present invention with utilize the mizoribine assay method of HPLC to have equal accuracy.
[composition 1]
30mM kaliumphosphate buffer pH6.0
50mM KCl
10mM ATP(pH7)
5mM NAD
The 20mM magnesium chloride
0.05% TX-100
0.19U/ml the BthNK of embodiment 12 preparations
0.16U/ml the BsuIMPDH of embodiment 33 preparations
1mM DTT
30mM NaCl
[composition 2]
100mM Tris/HCl pH of buffer 9.0
50mM KCl
0.05% TX-100
1mM DTT
20mM inosine 5 ' one phosphoric acid
[embodiment 53]
The comparison of<the first enzyme 〉
Use the BsuIMPDH of embodiment 33 preparations, first enzyme that derives from Methanococcus jannaschii or first enzyme that derives from the ancient green-ball bacterium of flicker of embodiment 3 preparations or the BthNK of embodiment 12 preparations of embodiment 2 preparations, make following [composition], measure the mizoribine of 1 μ g/ml.Use the automatic analytical engine of Hitachi's 7080 types in the mensuration.Parameter is as follows: sample is that 5 μ l, R1 are 100 μ l[compositions 1], R2 is 50 μ l[compositions 2], mensuration predominant wavelength is that 340nm, mensuration commplementary wave length are 405nm, condition determination is rate method A, reaction in 10 minutes, 20-22.
Its result is as follows: use under the situation of first enzyme that derives from Methanococcus jannaschii of embodiment 2 preparations, obtained about 80mAbs/ minute (9.3mAbs/ minute/mg) sensitivity; Use under the situation of first enzyme that derives from the ancient green-ball bacterium of flicker of embodiment 3 preparations, obtained about 80mAbs/ minute (16.7mAbs/ minute/mg) sensitivity; Use under the situation of BthNK of embodiment 12 preparations, obtained about 200mAbs/ minute (100mAbs/ minute/mg) sensitivity.By this result as can be known, the BthNK of embodiment 12 preparations is more than 10.7 times of first enzyme of embodiment 2 preparations, more than 6 times of first enzyme of embodiment 3 preparations to the efficient of the phosphorylation of mizoribine at least.In addition, take the result of this result and embodiment 4 into consideration, the BthNK of supposition embodiment 12 preparations is more than 21 times of adenosine kinase that derive from the adenosine kinase of mouse or derive from the people to the efficient of the phosphorylation of mizoribine.Therefore, though derive from first enzyme in the assay method that first enzyme of the ancient green-ball bacterium of flicker, first enzyme that derives from Methanococcus jannaschii, the adenosine kinase that derives from mouse, the adenosine kinase that derives from the people all can be used as mizoribine of the present invention and/or Ribavirin, but need to increase the consumption of this enzyme usually, so BthNK is suitable as first enzyme of the present invention most.
[composition 1]
30mM kaliumphosphate buffer pH7.0
75mM KCl
5mM ATP(pH7)
The 5mM magnesium chloride
1mM inosine 5 ' one phosphoric acid
1mM DTT
8.6mg/ml first enzyme of embodiment 2 preparation or
4.8mg/ml first enzyme of embodiment 3 preparation or
2.0mg/ml the BthNK of embodiment 12 preparations
[composition 2]
50mM Tris/HCl pH of buffer 8.5
75mM KCl
10mM NAD
1mM DTT
0.3U/ml the BsuIMPDH of embodiment 33 preparations
[embodiment 54]
The variant of<making second enzyme 〉
Make pHSG399/BsuIMPDH with the method identical with embodiment 28, embodiment 29 and embodiment 31.With this pHSG399/BsuIMPDH is template, use the primer of sequence table sequence numbering 25, sequence table sequence numbering 26, sequence table sequence numbering 27 and sequence table sequence numbering 28, conventional method by using PCR or the method identical with embodiment 28, embodiment 29 and embodiment 31, preparation pHSG399/BsuIMPDH[V432I, K449I], it is the plasmid of base sequence that contains the 2 amino acid variation bodies of the BsuIMPDH that encodes.Use the primer of sequence table sequence numbering 29, sequence table sequence numbering 30, sequence table sequence numbering 31, sequence table sequence numbering 32, sequence table sequence numbering 33, sequence table sequence numbering 34, sequence table sequence numbering 35, sequence table sequence numbering 36, sequence table sequence numbering 37 and sequence table sequence numbering 38, similarly make pHSG399/BsuIMPDH[D29N, S33Y, T158S, T270A, V387E], it is the plasmid of base sequence that contains the 5 amino acid variation bodies of the BsuIMPDH that encodes.Similarly make pHSG399/BsuIMPDH K345N and pHSG399/BsuIMPDH L367I, they are the plasmids of base sequence that contain the 1 amino acid variation body of the BsuIMPDH that encodes.Each transformant that will obtain by the method identical with embodiment 31 is inoculated in the LB nutrient culture media that contains 34 μ g/ml chloromycetin and 1mMIPTG, cultivates 1 day at 30 ℃.Its result, pHSG399/BsuIMPDH, pHSG399/BsuIMPDH[V432I, K449I], pHSG399/BsuIMPDH K345N, pHSG399/BsuIMPDH[D29N, S33Y, T158S, T270A, V387E] and the cultivation of the transformant of pHSG399/BsuIMPDH L367I to tire be respectively 0.64U/ml, 1.57U/ml, 2.69U/ml, 2.82U/ml and 3.09U/ml.Replace by these, in the manufacture method of second enzyme of the present invention, improved the turnout of second enzyme of the present invention.
[embodiment 55]
The mensuration of the mizoribine the in<red blood cell 〉
In the whole blood of 5ml heparin blood sampling, add the mizoribine of 10 μ g/ml, cultivate at 37 degree.After cultivating beginning, the whole blood in the time shown in the table 5, each 0.5ml of collection cultivated, and be blood plasma and haemocyte with its centrifuging.Haemocyte is suspended in the 0.5ml distilled water after cleaning 3 times with physiological saline, carries out ultrasonic disruption, filters with Ultrafree-MC Centrifugal Filter UnitsBiomax-10.Utilize the method identical to measure the concentration of the mizoribine in the resulting blood plasma and the concentration of the mizoribine in the filtrate with embodiment 52.The results are shown in Table 5, realized the mensuration of the mizoribine in the erythrocyte.
[table 5]
Incubation time (hour) | Before being about to add | 0.5 | 1 | 2 | 4 | 6 | 22 |
Mizoribine concentration in the blood plasma (μ g/ml) | -0.06 | 4.84 | 4.85 | 4.84 | 4.84 | 4.83 | 4.79 |
Mizoribine concentration in the filtrate (containing haemocyte) (μ g/ml) | -0.19 | 4.64 | 3.43 | 3.50 | 3.65 | 3.31 | 3.96 |
[embodiment 56]
The mensuration of the mizoribine the in<leucocyte 〉
Mouse lymph lymphoma L5178Y cell is cultivated in the nutrient culture media of being made up of 500ml DMEM (GIBCO), FBS (GIBCO), 100U penicillin, 100 μ g/ml streptomysins, and making cell suspending liquid concentration is 3 * 10
6Individual/ml.The mizoribine that adds 5 μ g/ml or 10 μ g/ml was therein further cultivated 3 hours or 18 hours at 37 ℃.The 20ml medium centrifugal is separated into the centrifugal cleer and peaceful cell of.Cell is suspended in the distilled water of 0.5ml after using the PBS of 1ml to clean 3 times, carries out ultrasonic disruption, filters with Ultrafree-MC Centrifugal Filter Units Biomax-10.Measure the concentration of the mizoribine in the resulting centrifugal supernatant and the concentration of the mizoribine in the filtrate with the method identical with embodiment 52.The results are shown in Table 6, realized the mensuration of the mizoribine in the leucocyte.
[table 6]
Incubation time (hour) | 3 hours | 3 hours | 3 hours | 18 hours | 18 hours | 18 hours |
Mizoribine addition (μ g/ml) | 0 | 5 | 10 | 0 | 5 | 10 |
Mizoribine concentration in the centrifugal supernatant (μ g/ml) | 0.04 | 3.69 | 7.75 | 0.02 | 3.60 | 8.08 |
Mizoribine concentration in the clasmatosis filtrate (μ g/ml) | 0.05 | 0.03 | 0.04 | 0.05 | 0.13 | 0.20 |
[embodiment 57]
The upright guest's of phosphoimidazole in the<leucocyte mensuration 〉
In the concentration identical with embodiment 56 is 3 * 10
6Add the mizoribine of concentration shown in the table 8 in the mouse lymph lymphoma L5178Y cell suspending liquid of individual/ml, further cultivated 4 hours at 37 ℃.Utilize the method identical to obtain clasmatosis filtrate with embodiment 56.Utilize the method identical to measure the concentration of the mizoribine of resulting filtrate, use the method identical to measure the concentration that phosphoimidazole stands the guest with embodiment 51 with embodiment 52.The concentration determination of mizoribine the results are shown in Table 7, and the upright guest's of phosphoimidazole concentration determination the results are shown in Table 8.Realized that mizoribine and the phosphoimidazole in the leucocyte founds guest's mensuration.
[table 7]
Mizoribine addition (μ g/ml) | 0 | 10 | 20 | 40 | 60 | 80 | 100 | 150 | 200 |
Mizoribine concentration in the clasmatosis filtrate (μ g/ml) | 0.1 | 2 | 5 | 6 | 10 | 10 | 18 | 24 | 27 |
[table 8]
Mizoribine addition (μ g/ml) | 0 | 10 | 20 | 40 | 60 | 80 | 100 | 150 | 200 |
Phosphoimidazole in the clasmatosis filtrate founds guest's concentration (μ g/ml) | 0.18 | 0.29 | 0.35 | 0.33 | 0.40 | 0.35 | 0.44 | 0.45 | 0.47 |
[embodiment 58]
The repeatability of<assay method of the present invention 〉
Select 10 corpse or other object for laboratory examination and chemical testing arbitrarily 47 corpse or other object for laboratory examination and chemical testing of serum that collect by the patient who uses among the embodiment 52, measure the repeatability (n=5) of assay method of the present invention from using mizoribine.The assay method of mizoribine is undertaken by the method identical with embodiment 52.Mean value, 2S.D. (standard deviation), CV (%) (coefficient of alteration) have been provided in the table 9.By this table as can be known, CV (%) is 1% only when big, and mizoribine assay method of the present invention is the good method of repeatability.
[table 9]
Corpse or other object for laboratory examination and chemical testing No. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
On average (μ g/ml) | 2.47 | 4.51 | 2.47 | 4.15 | 1.14 | 2.91 | 3.43 | 4.71 | 5.80 | 1.21 |
2S.D. | 0.05 | 0.03 | 0.04 | 0.04 | 0.02 | 0.06 | 0.02 | 0.03 | 0.01 | 0.01 |
CV(%) | 1.09 | 0.33 | 0.75 | 0.05 | 0.89 | 1.03 | 0.34 | 0.28 | 0.08 | 0.62 |
[embodiment 59]
The making of<standard reagent 〉
Dissolve mizoribine in the aqueous solution that contains 3.75% sucrose, 0.5% sweet mellow wine and 4.5%NaCl, making concentration is about 25 μ g/ml, 15 μ g/ml or 5 μ g/ml, makes the freeze drying mother liquor of standard reagent.Each 0.6ml ground of these mother liquors is injected freeze drying glass-tube bottle respectively, carry out freeze drying, make standard reagent with conventional method.This standard reagent uses after with the 3ml dissolved in distilled water.With the mizoribine concentration in the HPLC method bioassay standard reagent, the result is as follows: be that the calibration solution that the freeze drying of 25 μ g/ml is made with mother liquor is 4.90 μ g/ml, is that the calibration solution that the freeze drying of 15 μ g/ml is made with mother liquor is 3.40 μ g/ml, is that the calibration solution that the freeze drying of 5 μ g/ml is made with mother liquor is 1.01 μ g/ml with concentration with concentration with concentration.
[embodiment 60]
The day interpolation and poor in the daytime of<assay method of the present invention 〉
Measure the standard reagent of making among the embodiment 59 with the method identical, measure the day interpolation and poor in the daytime of assay method of the present invention with embodiment 52.Day interpolation is being by calculating with in a few days measuring 5 times, poorly in the daytime calculating with in a few days measuring 1 time by continuous 5 days.The result is as shown in table 10, and the mean value of the CV (%) of day interpolation is 0.64%, the mean value of poor CV (%) is 1.67% in the daytime.It is reported, J.Chromato., the HPLC method of 432 volumes, record in 340 pages, 1988 day interpolation and poor in the daytime be 2.503% and 3.898%, so to be the HPLC method equal above can carry out method for measuring with good accuracy for mizoribine assay method of the present invention.
[table 10]
Utilizability on the industry
The invention provides the straightforward procedure of measuring mizoribine and/or Ribavirin.
Sequence table
<110〉Asahi Kasei Pharma Corp
<120〉assay method of mizoribine and/or Ribavirin
<130>X1071061
<140>
<141>
<160>38
<210>1
<211>312
<212>PRT
<213〉Thailand's bulkholderia cepasea DSM13276
<400>1
<210>2
<211>939
<212>DNA
<213〉Thailand's bulkholderia cepasea DSM13276
<221>
<222>(0001)..(936)
<400>2
<210>3
<211>30
<212>DNA
<213〉artificial sequence
<400>3
<210>4
<211>33
<212>DNA
<213〉artificial sequence
<400>4
<210>5
<211>15
<212>PRT
<213〉the unknown
<223〉Xaa represents amino acid arbitrarily
<400>5
<210>6
<211>12
<212>PRT
<213〉the unknown
<223〉Xaa represents amino acid arbitrarily
<400>6
<210>7
<211>487
<212>PRT
<213〉hay bacillus ATCC23867
<223>
<400>7
<210>8
<211>1467
<212>DNA
<213〉hay bacillus ATCC23867
<221>
<222>(0001)..(1464)
<400>8
<210>9
<211>489
<212>PRT
<213>Oceanobacillus iheyensis DSM14731
<223>
<400>9
<210>10
<211>1470
<212>DNA
<213>Oceanobacillus iheyensis DSM14731
<221>
<222>(0001)..(1467)
<400>10
<210>11
<211>10
<212>PRT
<213〉the unknown
<400>11
<210>12
<211>10
<212>PRT
<213〉the unknown
<400>12
<210>13
<211>25
<212>DNA
<213〉artificial sequence
<400>13
<210>14
<211>25
<212>DNA
<213〉artificial sequence
<400>14
<210>15
<211>31
<212>DNA
<213〉artificial sequence
<400>15
<210>16
<211>33
<212>DNA
<213〉artificial sequence
<400>16
<210>17
<211>30
<212>DNA
<213〉artificial sequence
<400>17
<210>18
<211>29
<212>DNA
<213〉artificial sequence
<400>18
<210>19
<211>41
<212>DNA
<213〉artificial sequence
<400>19
<210>20
<211>30
<212>DNA
<213〉artificial sequence
<400>20
<210>21
<211>41
<212>DNA
<213〉artificial sequence
<400>21
<210>22
<211>30
<212>DNA
<213〉artificial sequence
<400>22
<210>23
<211>11
<212>PRT
<213〉the unknown
<400>23
<210>24
<211>17
<212>PRT
<213〉the unknown
<400>24
<210>25
<211>43
<212>DNA
<213〉artificial sequence
<400>25
<210>26
<211>43
<212>DNA
<213〉artificial sequence
<400>26
<210>27
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>28
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>29
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>30
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>31
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>32
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>33
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>34
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>35
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>36
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>37
<211>43
<212>DNA
<213〉artificial sequence
<400>
<210>38
<211>43
<212>DNA
<213〉artificial sequence
<400>
Claims (19)
1, the assay method of a kind of mizoribine and/or Ribavirin, it comprises following (1) operation:
(1) comprises the operation of first reaction, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine and/or the Ribavirin that may contain in the sample carried out phosphorylation mizoribine and/or Ribavirin.
2, the assay method of mizoribine as claimed in claim 1 and/or Ribavirin, wherein, described method comprises following (1), (2) and (3) each operation:
(1) comprises the operation of first reaction, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine and/or the Ribavirin that may contain in the sample carried out phosphorylation mizoribine and/or Ribavirin;
(2) suppress second operation of reacting, wherein, can catalysis second reaction second enzyme in the presence of, when carrying out second reaction, upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin that above-mentioned first reaction is generated contact with second enzyme, make second reaction be subjected to the inhibition of the degree corresponding with the amount of upright guest of this phosphoimidazole and/or phosphoric acid Ribavirin, and described second reaction is and the different reaction of above-mentioned first reaction, be following<a 〉~<c in any one reaction
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses,
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses,
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin; With
(3) detect the operation that second reaction is suppressed degree.
3, a kind of assay method of mizoribine, wherein, described method comprises following (1), (2) and (3) each operation:
(1) comprises first operation of reacting, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the mizoribine that may contain in the sample is carried out phosphorylation mizoribine;
(2) suppress second operation of reacting, wherein, can catalysis this second reaction second enzyme in the presence of, when carrying out second reaction, the upright guest of this phosphoimidazole that above-mentioned first reaction is generated contacts with second enzyme, makes second reaction be subjected to the inhibition of the degree corresponding with the upright guest's of this phosphoimidazole amount, and described second reaction is and the different reaction of above-mentioned first reaction, it is not suppressed by mizoribine, but the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses; With
(3) detect the operation that second reaction is suppressed degree.
4, a kind of assay method of Ribavirin, wherein, described method comprises following (1), (2) and (3) each operation:
(1) comprises first operation of reacting, in described first reaction, in the presence of first enzyme that can carry out phosphorylation, the Ribavirin that may contain in the sample is carried out phosphorylation Ribavirin;
(2) suppress second operation of reacting, wherein, can catalysis this second reaction second enzyme in the presence of, when carrying out second reaction, this phosphoric acid Ribavirin that above-mentioned first reaction is generated contacts with second enzyme, makes second reaction be subjected to the inhibition of the degree corresponding with the amount of this phosphoric acid Ribavirin, and described second reaction is and the different reaction of above-mentioned first reaction, it is not suppressed by Ribavirin, but the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses; With
(3) detect the operation that second reaction is suppressed degree.
5, as each described assay method of claim 1~4, wherein, first enzyme is from Thailand's bulkholderia cepasea.
6, as each described assay method of claim 2~5, wherein, second enzyme is inosine a 5 ' monophosphate dehydrogenase.
7, as each described assay method of claim 2~6, wherein, second enzyme is from hay bacillus or Oceanobacillus iheyensis.
8, the phosphorylation method of a kind of mizoribine and/or Ribavirin, this method is used any one enzyme in following (1)~(3):
(1) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine and/or Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction or makes Ribavirin become any one reaction at least in the reaction of phosphoric acid Ribavirin in the presence of phosphodonor;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
9, a kind of phosphorylation method of mizoribine, this method is used any one enzyme in following (1)~(3):
(1) can carry out the enzyme of phosphorylation to mizoribine, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to mizoribine, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of phosphodonor, catalysis makes mizoribine become the upright guest's of phosphoimidazole reaction;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
10, a kind of phosphorylation method of Ribavirin, this method is used any one enzyme in following (1)~(3):
(1) can carry out the enzyme of phosphorylation to Ribavirin, its amino acid sequence by sequence table sequence numbering 1 constitutes;
(2) can carry out the enzyme of phosphorylation to Ribavirin, it constitutes by lacking in the amino acid sequence of sequence table sequence numbering 1, replacing or added one or several amino acid whose amino acid sequence, and this amino acid sequence contains the amino acid sequence of ordered list sequence numbering 5 and the amino acid sequence of sequence table sequence numbering 6;
(3) have following<1 〉~<4 the enzyme of physicochemical property,
<1〉effect
At least in the presence of phosphodonor, catalysis makes Ribavirin become the reaction of phosphoric acid Ribavirin;
<2〉optimal pH
pH5.5~6.5;
<3〉pH stability
Scope in pH6~10 under 37 ℃, 3 hours condition keeps the activity more than 70%;
<4〉thermal stability
In the aqueous solution of the pH7.0 of 100mM kaliumphosphate buffer, when carrying out 20 minutes thermal treatment for 50 ℃, keep the activity more than 80%.
11, a kind of mensuration composition, it contains following compositions:
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to mizoribine and/or Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, its can catalysis second reaction, described second reaction is and the different reaction of above-mentioned first reaction, is following<a 〉~<c in any one reaction,
<a〉not suppressed by mizoribine, but the reaction that the upright guest of phosphoimidazole who generated by above-mentioned first reaction suppresses,
<b〉not suppressed by Ribavirin, but the reaction that the phosphoric acid Ribavirin that generated by above-mentioned first reaction suppresses,
<c〉the two suppresses not to be subjected to mizoribine and Ribavirin, but be subjected to the upright guest of phosphoimidazole that above-mentioned first reaction generates and the reaction of the two inhibition of phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
12, composition as claimed in claim 11, wherein, said composition is that the mizoribine mensuration that contains following compositions is used composition, described composition is:
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to mizoribine;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, its catalysis and the different reaction of above-mentioned first reaction, it is not suppressed by mizoribine, but suppressed by the upright guest of phosphoimidazole;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
13, composition as claimed in claim 11, wherein, said composition is that the Ribavirin mensuration that contains following compositions is used composition, described composition is:
(A1) first enzyme, it can catalysis carry out first reaction of phosphorylation to Ribavirin;
(A2) phosphodonor;
(A3) metallic ion;
(A4-1) second enzyme, its catalysis and the different reaction of above-mentioned first reaction, it is not suppressed by Ribavirin, but suppressed by the phosphoric acid Ribavirin;
(A4-2) inosine 5 ' one phosphoric acid; And
(A4-3) NAD (P) class.
14, as each described composition of claim 11~13, wherein, measure with in the composition at this, before being about to mensuration, add more than any one among following 3 kinds of compositions:
(A4-1) second enzyme,
(A4-2) inosine 5 ' one phosphoric acid and
(A4-3) NAD (P) class.
15,, wherein, can catalysis carry out first first enzyme that reacts of phosphorylation from Thailand's bulkholderia cepasea to mizoribine and/or Ribavirin as each described composition of claim 11~14.
16, as each described composition of claim 11~15, wherein, can catalysis second enzyme of second reaction be inosine 5 ' monophosphate dehydrogenase.
17, as each described composition of claim 11~16, wherein, can catalysis second enzyme of second reaction from hay bacillus or Oceanobacillus iheyensis.
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CN111474263A (en) * | 2020-04-27 | 2020-07-31 | 哈尔滨医科大学 | Pretreatment kit and detection method for rapid detection of ribavirin in human red blood cells |
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CN114062557B (en) * | 2021-11-19 | 2023-07-25 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Detection method for degradation impurities in mizoribine bulk drug |
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ATE290387T1 (en) * | 2001-12-21 | 2005-03-15 | Biopartners Gmbh | RIBAVIRIN GRANULES FOR THE PRODUCTION OF FILM TABLETS |
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CN111474263A (en) * | 2020-04-27 | 2020-07-31 | 哈尔滨医科大学 | Pretreatment kit and detection method for rapid detection of ribavirin in human red blood cells |
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